Streptococcus pneumoniae capsular polysaccharides and conjugates thereof

ABSTRACT

The invention relates to activated  Streptococcus pneumoniae  serotype 10A, 22F or 33F polysaccharides and processes for their preparation. The invention also relates to immunogenic conjugates comprising  Streptococcus pneumoniae  serotype 10A, 22F or 33F polysaccharides covalently linked to a carrier protein, processes for their preparation and immunogenic compositions and vaccines comprising them.

FIELD OF THE INVENTION

The invention relates to activated Streptococcus pneumoniae serotype10A, 22F or 33F polysaccharides and processes for their preparation. Theinvention also relates to immunogenic conjugates comprisingStreptococcus pneumoniae serotype 10A, 22F or 33F polysaccharidescovalently linked to a carrier protein, processes for their preparationand immunogenic compositions and vaccines comprising them.

BACKGROUND

Streptococcus pneumoniae are Gram-positive, lancet shaped cocci that areusually seen in pairs (diplococci), but also in short chains or assingle cells. They grow readily on blood agar plates with glisteningcolonies and display alpha hemolysis unless grown anaerobically wherethey show beta hemolysis. The cells of most pneumococcal serotypes havea capsule which is a polysaccharide coating surrounding each cell. Thiscapsule is a determinant of virulence in humans, as it interferes withphagocytosis by preventing antibodies from attaching to the bacterialcells. Currently there are more than 90 known pneumococcal capsularserotypes identified, with the 23 most common serotypes accounting forapproximately 90% of invasive disease worldwide. As a vaccine, thepneumococcal polysaccharide coat can confer a reasonable degree ofimmunity to Streptococcus pneumoniae in individuals with developed orunimpaired immune systems, but the capsular polysaccharide conjugated toa suitable carrier protein allows for an immune response in infants andelderly who are also at most risk for pneumococcal infections.

Since the introduction of the first 7-valent pneumococcal conjugatevaccine (PCV7 or Prevnar) in 2000, invasive disease from those sevenserotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) has nearly disappeared. Theaddition of serotypes 1, 3, 5, 6A, 7F and 19A in Prevnar 13 furtherdecreased the numbers of invasive pneumococcal disease.

None of the currently marketed pneumococcal vaccine provides anappropriate protection against serotype 10A, 22F or 33F Streptococcuspneumoniae in human and in particular in children less than 2 years old.Therefore, there is a need for immunogenic compositions that can be usedto induce an immune response against serotype 10A, 22F or 33FStreptococcus pneumonia.

SUMMARY OF THE INVENTION

In one aspect the present disclosure provides a process for preparing anactivated Streptococcus pneumoniae serotype 10A, 22F or 33F capsularpolysaccharide, the process comprising the steps of:

(a) reacting an isolated serotype 10A, 22F or 33F capsularpolysaccharide with an oxidizing agent; and;(b) quenching the oxidation reaction by addition of a quenching agentresulting in an activated Streptococcus pneumoniae serotype 10A, 22F or33F polysaccharide.

In a further aspect, the invention relates to activated Streptococcuspneumoniae serotype 10A, 22F or 33F capsular polysaccharide obtained orobtainable by the activation process disclosed herein.

In a further aspect, the present disclosure provides a process for thepreparation of an immunogenic conjugate comprising Streptococcuspneumoniae serotype 10A, 22F or 33F polysaccharide covalently linked toa carrier protein, the process comprising the steps of:

(a) compounding an activated serotype 10A, 22F or 33F polysaccharideobtained or obtainable by a process disclosed herein with a carrierprotein; and,(b) reacting the compounded, activated serotype WA, 22F or 33Fpolysaccharide and carrier protein with a reducing agent to form aserotype 10A, 22F or 33F polysaccharide:carrier protein conjugate.

In a further aspect, the present disclosure provides serotype 10A, 22For 33F polysaccharide:carrier protein conjugate obtained or obtainableby the process disclosed herein.

In a further aspect, the present disclosure provides immunogeniccompositions and vaccines comprising immunogenic conjugate disclosedherein.

In a further aspect, the present disclosure a method of treating orpreventing a Streptococcus pneumoniae infection, disease or conditionassociated with serotype 10A, 22F or 33F Streptococcus pneumoniae in asubject, the method comprising the step of administering atherapeutically or prophylactically effective amount of an immunogeniccomposition or a vaccine disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of Streptococcus pneumoniae capsularpolysaccharide Serotype 10A Repeat Unit.

FIG. 2 shows the structure of Streptococcus pneumoniae capsularpolysaccharide Serotype 22F Repeat Unit.

FIG. 3 shows the structure of Streptococcus pneumoniae capsularpolysaccharide Serotype 33F Repeat Unit.

FIG. 4 shows the molecular weight of activated serotype 22Fpolysaccharide as a function of the amount of oxidizing agent at 23° C.with and without quenching step.

FIG. 5 shows the molecular weight of activated serotype 22Fpolysaccharide as a function of the amount of oxidizing agent at 4° C.with and without quenching step.

FIG. 6 shows the molecular weight of activated serotype 33Fpolysaccharide as a function of the amount of oxidizing agent at 2 to 8°C. with or without quenching step or at 23° C. without quenching step.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the Examples included herein. Unless defined otherwise,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which theinvention pertains. Although any methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention, certain preferred methods andmaterials are described herein. In describing the embodiments andclaiming the invention, certain terminology will be used in accordancewith the definitions set out below.

As used herein, the singular forms “a”, “an”, and “the” include pluralreferences unless indicated otherwise. Thus, for example, references to“the method” includes one or more methods, and/or steps of the typedescribed herein and/or which will become apparent to one of ordinaryskill in the art upon reading this disclosure.

As used herein, the term “molecular weight” of polysaccharide or ofcarrier protein-polysaccharide conjugate refers to molecular weightcalculated by size exclusion chromatography (SEC) combined withmultiangle laser light scattering detector (MALLS).

As used herein, the term “degree of oxidation” (DO) refers to the numberof sugar repeat units per aldehyde group generated when the isolatedpolysaccharide is activated with an oxidizing agent. The degree ofoxidation of a polysaccharide can be determined using routine methodsknown to the man skilled in the art.

It is noted that in this disclosure, terms such as “comprises,”“comprised,” “comprising,” “contains,” “containing” and the like canhave the meaning attributed to them in U.S. patent law; e.g., they canmean “includes,” “included,” “including” and the like. Such terms referto the inclusion of a particular ingredients or set of ingredientswithout excluding any other ingredients. Terms such as “consistingessentially of” and “consists essentially of” have the meaningattributed to them in U.S. patent law, e.g., they allow for theinclusion of additional ingredients or steps that do not detract fromthe novel or basic characteristics of the invention, i.e., they excludeadditional unrecited ingredients or steps that detract from the novel orbasic characteristics of the invention. The terms “consists of” and“consisting of” have the meaning ascribed to them in U.S. patent law;namely, that these terms are closed ended. Accordingly, these termsrefer to the inclusion of a particular ingredient or set of ingredientsand the exclusion of all other ingredients.

As used herein, the term “conjugates” or “glycoconjugates” as usedherein refers to a polysaccharide covalently conjugated to a carrierprotein. Glycoconjugates of the invention and immunogenic compositionscomprising them may contain some amount of free polysaccharide.

As used herein, the term “serotype 10A glycoconjugate” or “serotype 10Aconjugate” refers to an isolated Streptococcus pneumoniae serotype 10Acapsular polysaccharide covalently conjugated to a carrier protein.

As used herein, the term “serotype 22F glycoconjugate” or “serotype 22Fconjugate” refers to an isolated Streptococcus pneumoniae serotype 22Fcapsular polysaccharide covalently conjugated to a carrier protein.

As used herein, the term “serotype 33F glycoconjugate” or “serotype 33Fconjugate” refers to an isolated Streptococcus pneumoniae serotype 33Fcapsular polysaccharide covalently conjugated to a carrier protein.

As used herein, the term “serotype 10A polysaccharide” refers to aStreptococcus pneumoniae serotype 10A capsular polysaccharide.

As used herein, the term “serotype 22F polysaccharide” refers to aStreptococcus pneumoniae serotype 22F capsular polysaccharide.

As used herein, the term “serotype 33F polysaccharide” refers to aStreptococcus pneumoniae serotype 33F capsular polysaccharide.

As used herein, the term “free polysaccharide” means a capsularpolysaccharide that is not covalently conjugated to the carrier protein,but is nevertheless present in the capsular polysaccharide-carrierprotein conjugate composition. The free polysaccharide may benon-covalently associated with (i.e., non-covalently bound to, adsorbedto, or entrapped in or with) the polysaccharide-carrier proteinconjugate.

The percentage of free polysaccharide is measured after the finalpurification of the serotype 10A, 22F or 33F capsularpolysaccharide-carrier protein conjugate. Preferably it is measuredwithin 4 weeks after the final purification. It is expressed as apercentage of the total polysaccharide in the sample.

As used herein, “to conjugate,” “conjugated” and “conjugating” refer toa process whereby a Streptococcus pneumoniae capsular polysaccharide, iscovalently attached to a carrier protein.

The term “subject” refers to a mammal, including a human, or to a bird,fish, reptile, amphibian or any other animal. The term “subject” alsoincludes household pets or research animals. Non-limiting examples ofhousehold pets and research animals include: dogs, cats, pigs, rabbits,rats, mice, gerbils, hamsters, guinea pigs, ferrets, monkeys, birds,snakes, lizards, fish, turtles, and frogs. The term “subject” alsoincludes livestock animals. Non-limiting examples of livestock animalsinclude: alpaca, bison, camel, cattle, deer, pigs, horses, llamas,mules, donkeys, sheep, goats, rabbits, reindeer, yak, chickens, geese,and turkeys.

In the preparation of multivalent conjugate pneumococcal vaccinesdirected to the prevention of invasive diseases caused by the organismStreptococcus pneumoniae (also known as pneumococcus), selectedStreptococcus pneumoniae serotypes are grown to supply polysaccharidesneeded to produce the vaccine. The cells are grown in fermentors withlysis induced at the end of the fermentation by addition of sodiumdeoxycholate or an alternate lysing agent. The lysate broth is thenharvested for downstream purification and the recovery of the capsularpolysaccharide which surrounds the bacterial cells. After activation andconjugation with a carrier protein, the polysaccharide is included inthe final vaccine product and confers immunity in the vaccine's targetpopulation to the selected Streptococcus pneumoniae serotypes.

The immunogenicity of a polysaccharide:carrier protein conjugate dependson several factors including the size of the polysaccharide.Polysaccharide size is a quality attribute assayed for in eachpreparation batch and must be appropriately controlled. High molecularweight capsular polysaccharides are able to induce certain antibodyimmune responses due to a higher valence of the epitopes present on theantigenic surface. It is generally preferable to prevent or to limitundesirable reduction of the size of the polysaccharide during thepreparation of the conjugate to retain the immunogenicity of thepolysaccharide. In addition, it is important to reduce the batch tobatch variability of the polysaccharide Molecular Weight during theactivation step and subsequent conjugation in order to maintain theconsistency of the quality attributes of the conjugate.

Reductive amination chemistry (RAC) has been demonstrated by theinventors as a suitable process for preparing the Streptococcuspneumoniae serotype 10A, 22F or 33F capsular polysaccharide-proteincarrier conjugate. The RAC approach involves activation of thepolysaccharide by oxidation and subsequent conjugation of the activatedpolysaccharide to a protein carrier by reduction. Serotype 10A, 33F and22F polysaccharides have proven to be particulary sensitivepolysaccharides and are susceptible to degradation and size reductionduring the oxidation step of the preparation of the conjugate. Inaddition, the use of hitherto known activation process producedactivated serotype 10A, 33F and 22F polysaccharide of variable molecularweights.

Therefore, there is an important need for a process for preparingactivated Streptococcus pneumoniae serotype 10A, 22F or 33Fpolysaccharides having a controlled and consistent molecular weight.

An object of the present invention is a process for preparing activatedStreptococcus pneumoniae serotype 10A, 22F or 33F polysaccharides. Inparticular, an object of the invention is a process for preparing anactivated Streptococcus pneumoniae serotype 10A, 22F or 33Fpolysaccharide, said process comprising the steps of:

(a) reacting isolated serotype 10A, 22F or 33F polysaccharide with anoxidizing agent; and,(b) quenching the oxidation reaction by addition of a quenching agentresulting in an activated Streptococcus pneumoniae serotype 10A, 22F or33F polysaccharide.

Isolation and Purification of Serotype 10A, 22F or 33F Polysaccharide

The structures of serotype 10A, 22F and 33F polysaccharides aredisclosed in the literature (see for example Kamerling J P C.Pneumococcal polysaccharides: a chemical view. In: Tomasz A, editor.Streptococcus pneumoniae, molecular biology and mechanisms of disease.New York, N.Y: Mary Ann Liebert, Inc.; 2000. pp. 81-114).

As shown in FIG. 1, the polysaccharide repeating unit of serotype 22Fconsists of a branched pentasaccharide backbone (one glucuronic acid(GIcpA), one glucopyranose (Glcp) and one galactofuranose (Galf) and tworhamnopyranoses (Rhap)) with a αGlcp branch linked to the C3 hydroxylgroup of βRhap 45. Approximately 80% of the C2 hydroxyl groups of theβRhap residue in the polysaccharide repeating unit are O-acetylated.

As shown in FIG. 2, the polysaccharide repeating unit of serotype 10Aconsists of a two D-galactosefuranose, three D-galactopyranose, oneN-acetylgalactosamine, and a phosphate bond linked with saccharide chainthrough one D-ribitol.

As shown in FIG. 3, the polysaccharide repeating unit of serotype 33Fconsists of three _(D)-galactopyranose, two D-galactofuranose, and oneD-glucopyranose. Notably, 5-D-galactofuranosyl residues are O-acetylatedin about 90% of 33F polysaccharide repeating units.

Serotype 10A, 22F and 33F capsular polysaccharides can be obtaineddirectly from bacteria using isolation procedures known to one ofordinary skill in the art (see for example methods disclosed U.S. PatentApp. Pub. Nos. 20060228380, 20060228381, 20070184071, 20070184072,20070231340, and 20080102498 or WO2008118752). In addition, they can beproduced using synthetic protocols.

Serotype 10A, 22F and 33F Streptococcus pneumoniae strains used to makethe respective polysaccharides that are used in the immunogenicconjugates of the invention may be obtained from established culturecollections or clinical specimens.

The purified serotype 10A, 22F and 33F polysaccharides are obtained bymethods well known to the man skilled in the art. The bacterial cellsare preferably grown in a soy based medium. Following fermentation ofbacterial cells that produce Streptococcus pneumoniae serotype 10A, 22For 33F capsular polysaccharides, the bacterial cells are lysed toproduce a cell lysate. The bacterial cells may be lysed using any lyticagent. A “lytic agent” is any agent that aids in cell wall breakdown andrelease of autolysin which causes cellular lysis including, for example,detergents. As used herein, the term “detergent” refers to any anionicor cationic detergent capable of inducing lysis of bacterial cells.Representative examples of such detergents for use within the methods ofthe present invention include deoxycholate sodium (DOC), N-lauroylsarcosine, chenodeoxycholic acid sodium, and saponins.

In one embodiment of the present invention, the lytic agent used forlysing bacterial cells is DOC. DOC is the sodium salt of the bile aciddeoxycholic acid, which is commonly derived from biological sources suchas cows or oxen. DOC activates the LytA protein, which is an autolysinthat is involved in cell wall growth and division in Streptococcuspneumoniae. The LytA protein has choline binding domains in itsC-terminal portion, and mutations of the lytA gene are known to produceLytA mutants that are resistant to lysis with DOC.

In one embodiment of the present invention, the lytic agent used forlysing bacterial cells is a non-animal derived lytic agent. Non-animalderived lytic agents for use within the methods of the present inventioninclude agents from non-animal sources with modes of action similar tothat of DOC (i. e., that affect LytA function and result in lysis ofStreptococcus pneumoniae cells). Such non-animal derived lytic agentsinclude, but are not limited to, analogs of DOC, surfactants,detergents, and structural analogs of choline. In one embodiment, thenon-animal derived lytic agent is selected from the group consisting ofdecanesulfonic acid, tert-octylphenoxy poly(oxyethylene)ethanols (e.g.Igepal® CA-630, CAS #: 9002-93-1, available from Sigma Aldrich, St.Louis, Mo.), octylphenol ethylene oxide condensates (e.g. Triton® X-100,available from Sigma Aldrich, St. Louis, Mo.), N-lauroyl sarcosinesodium, lauryl iminodipropionate, sodium dodecyl sulfate,chenodeoxycholate, hyodeoxycholate, glycodeoxycholate,taurodeoxycholate, taurochenodeoxycholate, and cholate. In anotherembodiment, the non-animal derived lytic agent is N-lauroyl sarcosine.In another embodiment, the lytic agent is N-lauroyl sarcosine sodium.

The capsular polysaccharides may then be purified from the cell lysateusing purification techniques known in the art, including the use ofcentrifugation, depth filtration, precipitation, ultra-filtration,treatment with activate carbon, diafiltration and/or columnchromatography (See, for example, U.S. Patent App. Pub. Nos.20060228380, 20060228381, 20070184071, 20070184072, 20070231340, and20080102498 or WO2008118752). The purified serotype 10A, 22F or 33Fpolysaccharides can then be used for the preparation of immunogenicconjugates.

Preferably, in order to generate serotype 22F conjugates or serotype 33Fconjugates with advantageous filterability characteristics and/oryields, sizing of the polysaccharide to a lower molecular weight (MVV)range is performed prior to the conjugation to a carrier protein.Advantageously, the size of the purified serotype 22F polysaccharide orserotype 33F polysaccharide is reduced while preserving criticalfeatures of the structure of the polysaccharide such as for example thepresence of O-acetyl groups. Preferably, the size of the purifiedserotype 22F polysaccharide or serotype 33F polysaccharide is reduced bymechanical homogenization.

In a preferred embodiment, the size of the purified polysaccharide isreduced by high pressure homogenization. High pressure homogenizationachieves high shear rates by pumping the process stream through a flowpath with sufficiently small dimensions. The shear rate is increased byusing a larger applied homogenization pressure and exposure time can beincreased by recirculating the feed stream through the homogenizer.

The high pressure homogenization process is particularly appropriate forreducing the size of the purified serotype 22F polysaccharide orserotype 33F polysaccharide while preserving the structural features ofthe polysaccharide such as the presence of O-acetyl groups.

The isolated serotype 22F polysaccharide obtained by purification ofserotype 22F capsular polysaccharide from the Streptococcus pneumoniaelysate and optionally sizing of the purified polysaccharide can becharacterized by different parameters including for example themolecular weight, and the mM of acetate per mM of serotype 22Fpolysaccharide. In a preferred embodiment, the sized serotype 22Fpolysaccharide has a molecular weight comprised between 400 and 700 kDa.

The isolated serotype 33F polysaccharide obtained by purification ofserotype 33F capsular polysaccharide from the Streptococcus pneumoniaelysate and optionally sizing of the purified polysaccharide can becharacterized by different parameters including for example themolecular weight, and the mM of acetate per mM of serotype 33Fpolysaccharide.

The degree of O-acetylation of the polysaccharide can be determined byany method known in the art, for example, by proton NMR (Lemercinier andJones (1996) Carbohydrate Research 296; 83-96, Jones and Lemercinier(2002) J. Pharmaceutical and Biomedical Analysis 30; 1233-1247, WO05/033148 or WO00/56357. Another commonly used method is described inHestrin (1949) J. Biol. Chem. 180; 249-261. Preferably, the presence ofO-acetyl groups is determined by ion-HPLC analysis.

In a preferred embodiment, the isolated serotype 22F polysaccharide isobtained by a process comprising the steps of:

-   -   preparing a fermentation culture of serotype 22F Streptococcus        pneumonia bacterial cells;    -   lysing the bacterial cells in said fermentation culture; and,    -   purifying serotype 22F polysaccharide from the fermentation        culture.

In a preferred embodiment, the isolated serotype 22F polysaccharide isobtained by a process comprising the steps of:

-   -   preparing a fermentation culture of serotype 22F Streptococcus        pneumonia bacterial cells;    -   lysing the bacterial cells in said fermentation culture;    -   purifying serotype 22F polysaccharide from the fermentation        culture; and,    -   sizing the serotype 22F polysaccharide by mechanical sizing,        preferably by high pressure homogenization.

In a preferred embodiment, the isolated serotype 22F polysaccharide isobtained by a process comprising the steps of:

-   -   preparing a fermentation culture of serotype 22F Streptococcus        pneumonia bacterial cells;    -   lysing the bacterial cells in said fermentation culture;    -   purifying serotype 22F polysaccharide from the fermentation        culture; and,    -   sizing the serotype 22F polysaccharide by mechanical sizing,        preferably by high pressure homogenization,        wherein the isolated serotype 22F polysaccharide has a molecular        weight comprised between 400 and 700 kDa.

In a preferred embodiment, the isolated serotype 33F polysaccharide isobtained by a process comprising the steps of:

-   -   preparing a fermentation culture of serotype 33F Streptococcus        pneumonia bacterial cells;    -   lysing the bacterial cells in said fermentation culture; and,    -   purifying serotype 33F polysaccharide from the fermentation        culture.

In a preferred embodiment, the isolated serotype 33F polysaccharide isobtained by a process comprising the steps of:

-   -   preparing a fermentation culture of serotype 33F Streptococcus        pneumonia bacterial cells;    -   lysing the bacterial cells in said fermentation culture;    -   purifying serotype 33F polysaccharide from the fermentation        culture; and,    -   sizing the serotype 33F polysaccharide by mechanical sizing,        preferably by high pressure homogenization.

The isolated serotype 10A polysaccharide obtained by purification ofserotype 10A capsular polysaccharide from the Streptococcus pneumoniaelysate and optionally sizing of the purified polysaccharide can becharacterized by different parameters including for example themolecular weight. Preferably, the isolated serotype 10A polysaccharideis not sized.

In a preferred embodiment, the isolated serotype 10A polysaccharide isobtained by a process comprising the steps of:

-   -   preparing a fermentation culture of serotype 10A Streptococcus        pneumonia bacterial cells;    -   lysing the bacterial cells in said fermentation culture; and,    -   purifying serotype 10A polysaccharide from the fermentation        culture.

Activation of Serotype 10A, 22F or 33F Polysaccharide

The isolated serotype 10A, 22F or 33F polysaccharide is activated by aprocess comprising the step of:

(a) reacting an isolated serotype 10A, 22F or 33F polysaccharide with anoxidizing agent; and,(b) quenching the oxidation reaction by addition of a quenching agentresulting in an activated Streptococcus pneumoniae serotype 10A, 22F or33F polysaccharide.

In a preferred embodiment, the concentration of serotype 10A, 22F or 33Fpolysaccharide in step (a) is between 0.1 and 10 mg/mL. In a preferredembodiment, the concentration of serotype 10A, 22F or 33F polysaccharidein step (a) is between 0.5 and 5 mg/mL. In a preferred embodiment, theconcentration of serotype 10A, 22F or 33F polysaccharide in step (a) isbetween 1 and 3 mg/mL. In a preferred embodiment, the concentration ofserotype 22F or 33F polysaccharide in step (a) is about 2 mg/mL. In apreferred embodiment, the concentration of serotype 10A polysaccharidein step (a) is about 2.5 mg/mL.

In a preferred embodiment, the oxidizing agent is periodate. Theperiodate oxidises vicinal hydroxyl groups to form carbonyl or aldehydegroups and causes cleavage of a C—C bond. The term ‘periodate’ includesboth periodate and periodic acid. This term also includes bothmetaperiodate (IO₄ ⁻) and orthoperiodate (IO₆ ⁵⁻). The term ‘periodate’also includes the various salts of periodate including sodium periodateand potassium periodate. In a preferred embodiment, the oxidizing agentis sodium periodate. In a preferred embodiment, the periodate used forthe oxidation of serotype 10A, 22F or 33F polysaccharide ismetaperiodate. In a preferred embodiment the periodate used for theoxidation of serotype 10A, 22F or 33F polysaccharide is sodiummetaperiodate.

In a preferred embodiment, the polysaccharide is reacted with 0.01 to10, 0.05 to 5, 0.1 to 1, 0.5 to 1, 0.7 to 0.8, 0.01 to 0.2, 0.05 to 0.5,0.05 to 0.2, 0.1 to 0.3 molar equivalents of oxidizing agent. In apreferred embodiment, the polysaccharide is reacted with about 0.05,0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7,0.75, 0.8, In a preferred embodiment, the polysaccharide is reacted withabout 0.10 molar equivalent of oxidizing agent. In a preferredembodiment, the polysaccharide is reacted with about 0.15 molarequivalent of oxidizing agent. In a preferred embodiment, thepolysaccharide is reacted with about 0.25 molar equivalent of oxidizingagent. In a preferred embodiment, the polysaccharide is reacted withabout 0.5 molar equivalent of oxidizing agent. In a preferredembodiment, the polysaccharide is reacted with about 0.8 molarequivalent of oxidizing agent.

In a preferred embodiment, the duration of the reaction in step (a) isbetween 1 and 50, 1 and 40, 1 and 30, 1 and 25, 1 and 20, 1 and 10, 10to 50, 10 to 40, 10 to 30, 10 to 20 hours. In a preferred embodiment,when the polysaccharide is serotype 10A polysaccharide, the duration ofthe reaction in step (a) is between 1 to 10 hours. In a preferredembodiment, when the polysaccharide is serotype 10A polysaccharide, theduration of the reaction in step (a) is about 1, 2, 3, 4, 5, 6, 7, 8, 9,10 hours. In a preferred embodiment, when the polysaccharide is serotype10A polysaccharide, the duration of the reaction in step (a) is about 4hours. In a preferred embodiment, when the polysaccharide is serotype22F polysaccharide, the duration of the reaction in step (a) is between10 and 20 hours. In a preferred embodiment, when the polysaccharide isserotype 22F polysaccharide, the duration of the reaction in step (a) isabout 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 hours. In a preferredembodiment, when the polysaccharide is serotype 33F polysaccharide, theduration of the reaction in step (a) is between 15 to 25 hours. In apreferred embodiment, when the polysaccharide is serotype 33Fpolysaccharide, the duration of the reaction in step (a) is about 15,16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 hours. In a preferredembodiment, when the polysaccharide is serotype 33F polysaccharide, theduration of the reaction in step (a) is about 20 hours.

In a preferred embodiment, the temperature of the reaction in step (a)is maintained between 1 to 30° C., 1 to 10° C., 10 to 20° C., 20 to 30°C., 2 to 8° C. In a preferred embodiment, the temperature of thereaction in step (a) is maintained at about 5° C.

In a preferred embodiment, step (a) is carried out in a buffer selectedfrom sodium phosphate, potassium phosphate,2-(N-morpholino)ethanesulfonic acid (MES) or Bis-Tris. In a preferredembodiment step (a) is carried out in potassium phosphate buffer. In apreferred embodiment step (a) is carried out in sodium phosphate buffer.

In a preferred embodiment the buffer has a concentration of between 1 to500 mM, 1 to 300 mM, 50 to 200 mM. In a preferred embodiment the bufferhas a concentration of about 100 mM.

In a preferred embodiment the pH in step (a) is between 4 and 8, 5 and7, 5.5 and 6.5. In a preferred embodiment the pH in step (a) is about 6.In a preferred embodiment the pH in step (a) is about 5.8.

In one embodiment, the quenching agent is selected from vicinal diols,1,2-aminoalcohols, amino acids, glutathione, sulfite, bisulfate,dithionite, metabisulfite, thiosulfate, phosphites, hypophosphites orphosphorous acid.

In one embodiment, the quenching agent is a 1,2-aminoalcohols of formula

where R¹ is selected from H, methyl, ethyl, propyl or isopropyl.

In one embodiment, the quenching agent is selected from sodium andpotassium salts of sulfite, bisulfate, dithionite, metabisulfite,thiosulfate, phosphites, hypophosphites or phosphorous acid.

In one embodiment, the quenching agent is an amino acid. In oneembodiment, said amino acid is selected from serine, threonine,cysteine, cystine, methionine, proline, hydroxyproline, tryptophan,tyrosine, and histidine.

In one embodiment, the quenching agent is a sulfite such as bisulfate,dithionite, metabisulfite, thiosulfate.

In one embodiment, the quenching agent is a compound comprising twovicinal hydroxyl groups (vicinal diols), i.e two hydroxyl groupscovalently linked to two adjacent carbon atoms.

Preferably, the quenching agent is a compound of formula (II)

wherein R¹ and R² are each independently selected from H, methyl, ethyl,propyl or isopropyl.

In a preferred embodiment, the quenching agent is glycerol, ethyleneglycol, propan-1,2-diol, butan-1,2-diol or butan-2,3-diol, ascorbicacid. In a preferred embodiment, the quenching agent is butan-2,3-diol.

In a preferred embodiment, the oxidizing agent is neutralized by theaddition of 0.1 to 10, 0.5 to 5, 0.5 to 3, 0.5 to 2 molar equivalents ofquenching agent. In a preferred embodiment, the oxidizing agent isneutralized by the addition of about 0.5, 1, 1.5, 2, 2.5, 3 molarequivalents of quenching agent. In a preferred embodiment, the oxidizingagent is neutralized by the addition of about 2 molar equivalents ofquenching agent.

In a preferred embodiment, the duration of step (b) is between 0.1 and10, 0.5 and 5, or 0.5 and 2 hours. In a preferred embodiment, theduration of step (b) is about 0.5, 1, 1.5, 2.5 or 3 hours.

In a preferred embodiment, the temperature of the reaction in step (b)is maintained between 1 and 30° C., 1 and 25° C., 1 and 20° C., 1 and10° C., 10 and 20° C., 2 and 8° C. In a preferred embodiment, thetemperature of the reaction in step (b) is maintained at about 1, 2, 3,4, 5, 6, 7 or 8° C.

In a preferred embodiment the pH in step (b) is between 4 and 8, 5 and7, 5.5 and 6.5. In a preferred embodiment the pH in step (a) is about 6.

In a preferred embodiment, the activated serotype 10A, 22F or 33Fpolysaccharide is purified. The activated serotype 10A, 22F or 33Fpolysaccharide can be purified according to methods known to the manskilled in the art such as gel permeation chromatography (GPC), dialysisor ultrafiltration/diafiltration. For example, the activatedpolysaccharide is purified by concentration and diafiltration using anultrafiltration device.

In preferred embodiment, the isolated serotype 10A polysaccharide isactivated by a process comprising the step of:

(a) reacting isolated serotype 10A polysaccharide with periodate; and,(b) quenching the oxidation reaction by addition of butan-2,3-diolresulting in an activated serotype 10A polysaccharide.

In preferred embodiment, the isolated serotype 10A polysaccharide isactivated by a process comprising the step of:

(a) reacting isolated serotype 10A polysaccharide with periodate at atemperature between 2 and 8° C.; and,(b) quenching the oxidation reaction by addition of butan-2,3-diol at atemperature between 2 and 8° C. resulting in an activated serotype WApolysaccharide.

In preferred embodiment, the isolated serotype 10A polysaccharide isactivated by a process comprising the step of:

(a) reacting isolated serotype 10A polysaccharide with 0.2 to 0.3 molarequivalent periodate at a temperature between 2 and 8° C.; and,(b) quenching the oxidation reaction by addition of 0.5 to 2 molarequivalent of butan-2,3-diol at a temperature between 2 and 8° C.resulting in an activated serotype 10A polysaccharide.

In preferred embodiment, the isolated serotype 22F polysaccharide isactivated by a process comprising the step of:

(a) reacting isolated serotype 22F polysaccharide with periodate; and,(b) quenching the oxidation reaction by addition of butan-2,3-diolresulting in an activated serotype 22F polysaccharide.

In preferred embodiment, the isolated serotype 22F polysaccharide isactivated by a process comprising the step of:

(a) reacting isolated serotype 22F polysaccharide with periodate at atemperature between 2 and 8° C.; and,(b) quenching the oxidation reaction by addition of butan-2,3-diol at atemperature between 2 and 8° C. resulting in an activated serotype 22Fpolysaccharide.

In preferred embodiment, the isolated serotype 22F polysaccharide isactivated by a process comprising the step of:

(a) reacting isolated serotype 22F polysaccharide with 0.05 to 0.2 molarequivalent periodate at a temperature between 2 and 8° C.; and,(b) quenching the oxidation reaction by addition of 2 to 3, preferably2, molar equivalents of butan-2,3-diol at a temperature between 2 and 8°C. resulting in an activated serotype 22F polysaccharide.

In preferred embodiment, the isolated serotype 33F polysaccharide isactivated by a process comprising the step of:

(a) reacting isolated serotype 33F polysaccharide with periodate; and,(b) quenching the oxidation reaction by addition of butan-2,3-diolresulting in an activated serotype 33F polysaccharide.

In preferred embodiment, the isolated serotype 33F polysaccharide isactivated by a process comprising the step of:

(a) reacting isolated serotype 33F polysaccharide with periodate at atemperature between 2 and 8° C.; and,(b) quenching the oxidation reaction by addition of butan-2,3-diol at atemperature between 2 and 8° C. resulting in an activated serotype 33Fpolysaccharide.

In preferred embodiment, the isolated serotype 33F polysaccharide isactivated by a process comprising the step of:

(a) reacting isolated serotype 33F polysaccharide with 0.05 to 0.2 molarequivalent periodate at a temperature between 2 and 8° C.; and,(b) quenching the oxidation reaction by addition of 0.5 to 1.5 molarequivalent of butan-2,3-diol at a temperature between 2 and 8° C.resulting in an activated serotype 33F polysaccharide.

In a preferred embodiment, the invention relates to an activatedserotype 10A capsular polysaccharide obtained or obtainable by the abovedisclosed process.

In a preferred embodiment, the invention relates to an activatedserotype 22F capsular polysaccharide obtained or obtainable by the abovedisclosed process.

In a preferred embodiment, the invention relates to an activatedserotype 33F capsular polysaccharide obtained or obtainable by the abovedisclosed process.

In a preferred embodiment the activated serotype 10A polysaccharideobtained from step (b) has a molecular weight between 20 and 100%, 30and 95%, 40 and 95%, 60 to 95%, 85 to 95%, or about 90%, of themolecular weight of the isolated polysaccharide used in step (a).

In a preferred embodiment the activated serotype 10A polysaccharideobtained from step (b) has a molecular weight of at least 20, 25, 30,35, 40 or 45% of the molecular weight of the isolated polysaccharideused in step (a). In a preferred embodiment the activated serotype 10Apolysaccharide obtained from step (b) has a molecular weight of at least50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of the molecularweight of the isolated polysaccharide used in step (a).

In a preferred embodiment the activated serotype 22F polysaccharideobtained from step (b) has a molecular weight between 50 and 100%, 60 to95%, 85 to 95% or about 90% of the molecular weight of the isolatedpolysaccharide used in step (a).

In a preferred embodiment the activated serotype 22F polysaccharideobtained from step (b) has a molecular weight of at least 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of the molecular weight ofthe isolated polysaccharide used in step (a).

In a preferred embodiment the activated serotype 33F polysaccharideobtained from step (b) has a molecular weight between 50 and 100%, 60 to95%, 85 to 95%, or about 90% of the molecular weight of the isolatedpolysaccharide used in step (a).

In a preferred embodiment the activated serotype 33F polysaccharideobtained from step (b) has a molecular weight of at least 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of the molecular weight ofthe isolated polysaccharide used in step (a).

In a preferred embodiment the degree of oxidation of the activatedserotype 10A, 22F or 33F polysaccharide is between 2 and 30, 2 and 25, 2and 20, 2 and 15, 2 and 10, 2 and 5, 5 and 30, 5 and 25, 5 and 20, 5 and15, 5 and 10, 10 and 30, 10 and 25, 10 and 20, 10 and 15, 5 and 25, 10and 30, 15 and 30, 15 and 25, 15 and 20, 20 to 30, 20 to 25. In apreferred embodiment the degree of oxidation of the activated serotype10A, 22F or 33F polysaccharide is between 2 and 10, 4 and 8, 4 and 6, 6and 8, 6 and 12, 8 and 14, 9 and 11, 10 and 16, 12 and 16, 14 and 18, 16and 20, 16 and 18, 18 and 22, 18 and 20.

In a preferred embodiment, the activated serotype 10A polysaccharide hasa molecular weight between 50 and 400, 50 and 350, 50 and 300, 50 and250, 50 and 200, 100 and 300, 100 and 250 or 100 and 200 kDa. In apreferred embodiment, the activated serotype 10A polysaccharide has amolecular weight between 50 and 300 kDa. In a preferred embodiment, theactivated serotype 10A polysaccharide has a molecular weight between 100and 200 kDa. In a preferred embodiment, the activated serotype 10Apolysaccharide has a molecular weight between 100 and 200 kDa and adegree of oxidation between 5 and 20, 5 and 15, 8 and 14, 8 and 12 or 9and 11. In a preferred embodiment, the activated serotype 10Apolysaccharide has a molecular weight between 100 and 200 kDa and adegree of oxidation between 9 and 11.

In a preferred embodiment, the activated serotype 22F polysaccharide hasa molecular weight between 25 and 1000, 100 and 1000, 300 and 800, 300and 700, 300 and 600, 400 and 1000, 400 and 800, 400 and 700 or 400 and600 kDa. In a preferred embodiment, the activated serotype 22Fpolysaccharide has a molecular weight between 300 and 800 kDa. In apreferred embodiment, the activated serotype 22F polysaccharide has amolecular weight between 400 and 800 kDa. In a preferred embodiment, theactivated serotype 22F polysaccharide has a molecular weight between 400and 600 kDa. In a preferred embodiment, the activated serotype 22Fpolysaccharide has a molecular weight between 400 and 800 kDa and adegree of oxidation between 10 and 25, 10 and 20, 12 and 20 or 14 and18. In a preferred embodiment, the activated serotype 22F polysaccharidehas a molecular weight between 400 and 800 kDa and a degree of oxidationbetween 10 and 20.

In a preferred embodiment, the activated serotype 22F polysaccharidecomprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7 or about 0.8 mMacetate per mM serotype 22F polysaccharide. In a preferred embodiment,the activated serotype 22F polysaccharide comprises at least 0.5, 0.6 or0.7 mM acetate per mM serotype 22F polysaccharide. In a preferredembodiment, the activated serotype 22F polysaccharide comprises at least0.6 mM acetate per mM serotype 22F polysaccharide. In a preferredembodiment, the activated serotype 22F polysaccharide comprises at least0.7 mM acetate per mM serotype 22F polysaccharide.

In a preferred embodiment, the activated serotype 22F polysaccharide hasa molecular weight between 400 and 800 kDa and comprises at least 0.6 mMacetate per mM serotype 22F polysaccharide.

In a preferred embodiment, the activated serotype 22F polysaccharide hasa molecular weight between 400 and 800 kDa, a degree of oxidationbetween 12 and 20 and comprises at least 0.6 mM acetate per mM serotype22F polysaccharide.

In a preferred embodiment, the activated serotype 33F polysaccharide hasa molecular weight between 50 and 1250, 200 and 1200, 500 and 1200, 500and 1000, 700 and 1200, 800 and 1200, 800 and 1100, 900 and 1200, 800and 1000 Da. In a preferred embodiment, the activated serotype 33Fpolysaccharide has a molecular weight between 500 and 1000 kDa. Inanother preferred embodiment, the activated serotype 33F polysaccharidehas a molecular weight between 50 and 600, 50 and 500 or 50 and 400 kDa.

In a preferred embodiment, the activated serotype 33F polysaccharidecomprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 or about 0.9mM acetate per mM serotype 33F polysaccharide. In a preferredembodiment, the activated serotype 33F polysaccharide comprises at least0.6, 0.7 or 0.8 mM acetate per mM serotype 33F polysaccharide. In apreferred embodiment, the activated serotype 33F polysaccharidecomprises at least 0.6 mM acetate per mM serotype 33F polysaccharide. Ina preferred embodiment, the activated serotype 33F polysaccharidecomprises at least 0.7 mM acetate per mM serotype 33F polysaccharide.

In a preferred embodiment, the activated serotype 33F polysaccharide hasa molecular weight between 800 and 1200 kDa and comprises at least 0.7mM acetate per mM serotype 33F polysaccharide. In a preferredembodiment, the activated serotype 33F polysaccharide has a molecularweight between 800 and 1200 kDa, comprises at least 0.7 mM acetate permM serotype 33F polysaccharide and has a degree of oxidation between 10and 20.

In a preferred embodiment, the activated serotype 33F polysaccharide hasa molecular weight between 50 and 200 kDa and comprises at least 0.7 mMacetate per mM serotype 33F polysaccharide.

In a preferred embodiment, the activated serotype 33F polysaccharide hasa molecular weight between 50 and 200 kDa, comprises at least 0.7 mMacetate per mM serotype 33F polysaccharide and has a degree of oxidationbetween 10 and 20.

In an embodiment, the activated serotype 10A, 22F or 33F polysaccharideis lyophilized, optionally in the presence of acryoprotectant/lyoprotectant. In a preferred embodiment, thecryoprotectant/lyoprotectant is selected from sucrose, trehalose,raffinose, stachyose, melezitose, dextran, mannitol, lactitol andpalatinit. In a preferred embodiment, the cryoprotectant/lyoprotectantis sucrose. The lyophilized activated polysaccharide can then becompounded with a solution comprising the carrier protein.

In another embodiment, the activated serotype 10A, 22F or 33Fpolysaccharide is compounded with the carrier protein and lyophilizedoptionally in the presence of a cryoprotectant/lyoprotectant. In apreferred embodiment, the cryoprotectant/lyoprotectant is selected fromsucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol,lactitol and palatinit. In a preferred embodiment, thecryoprotectant/lyoprotectant is sucrose. The co-lyophilizedpolysaccharide and carrier protein can then be resuspended in solutionand reacted with a reducing agent.

In an embodiment, the invention relates to a lyophilized activatedserotype 10A polysaccharide. In an embodiment, the invention relates toa lyophilized activated serotype 22F polysaccharide. In an embodiment,the invention relates to a lyophilized activated serotype 33Fpolysaccharide.

In an embodiment the invention relates to the co-lyophilized activatedserotype 10A polysaccharide and protein carrier. In a preferredembodiment, the protein carrier is CRM₁₉₇. In an embodiment theinvention relates to the co-lyophilized activated serotype 22Fpolysaccharide and protein carrier. In a preferred embodiment, theprotein carrier is CRM₁₉₇. In an embodiment the invention relates to theco-lyophilized activated serotype 33F polysaccharide and proteincarrier. In a preferred embodiment, the protein carrier is CRM₁₉₇.

Conjugation of Activated Serotype 10A, 22F or 33F Polysaccharide with aCarrier Protein

The activated serotype 10A, 22F or 33F polysaccharide disclosed hereincan be conjugated to a carrier protein by a process comprising the stepsof:

(a) compounding the activated serotype 10A, 22F or 33F polysaccharidewith a carrier protein; and,(b) reacting the compounded activated serotype 10A, 22F or 33Fpolysaccharide and carrier protein with a reducing agent to form aserotype 10A, 22F or 33F polysaccharide:carrier protein conjugate.

The conjugation of activated serotype 22F or 33F polysaccharide with aprotein carrier by reductive amination in dimethylsulfoxide (DMSO) issuitable to preserve the O-acetyl content of the polysaccharide ascompared for example to reductive amination in aqueous phase where thelevel of O-acetylation of the polysaccharide is significantly reduced.In a preferred embodiment, step (a) and step (b) are carried out inDMSO.

In a preferred embodiment, step (a) comprises dissolving lyophilizedserotype 10A, 22F or 33F polysaccharide in an aqueous solutioncomprising a carrier protein or in a solution comprising a carrierprotein and DMSO. In a preferred embodiment, step (a) comprisesdissolving co-lyophilized serotype 10A, 22F or 33F polysaccharide andcarrier protein in an aqueous solution or in DMSO.

When step (a) and (b) are carried out in an aqueous solution, saidsolution comprises a buffer, preferably selected from buffer, preferablyselected from PBS, MES, HEPES, Bis-tris, ADA, PIPES. MOPSO, BES, MOPS,DIPSO, MOBS, HEPPSO, POPSO, TEA, EPPS, Bicine or HEPB, at a pH between6.0 and 8.5, 7 and 8 or 7 and 7.5. In a preferred embodiment the bufferis PBS. In a preferred embodiment the pH is about 7.3.

In a preferred embodiment, the concentration of activated serotype 10A,22F or 33F polysaccharide in step (a) is between 0.1 and 10 mg/mL, 0.5and 5 mg/mL, 0.5 and 2 mg/mL. In a preferred embodiment, theconcentration of activated serotype 10A, 22F or 33F polysaccharide instep (a) is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9 or 3 mg/mL.

In a preferred embodiment the initial ratio (weight by weight) ofactivated serotype 10A, 22F or 33F polysaccharide to carrier protein isbetween 5:1 and 0.1:1, 2:1 and 0.1:1, 2:1 and 1:1, 1.5:1 and 1:1, 0.1:1and 1:1, 0.3:1 and 1:1, 0.6:1 and 1:1.2. In a preferred embodiment theinitial ratio of activated serotype 10A, 22F or 33F polysaccharide tocarrier protein is about 0.6:1 and 1:1.2. In a preferred embodiment theinitial ratio of activated serotype 10A, 22F or 33F polysaccharide tocarrier protein is about 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1,1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1.

In a preferred embodiment, in step (b), the activated serotype 10A, 22For 33F polysaccharide is reacted with between 0.1 and 10 molarequivalents, 0.5 and 5 molar equivalents 0.5 and 2.5 molar equivalentsof reducing agent. In a preferred embodiment, in step (b), the activatedserotype 10A, 22F or 33F is reacted with about 0.5, 0.6, 0.7, 0.8, 0.9,1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4 or2.5 molar equivalents of reducing agent.

In an embodiment, the reducing agent is sodium cyanoborohydride, sodiumtriacetoxyborohydride, sodium or zinc borohydride in the presence ofBronsted or Lewis acids, amine boranes such as pyridine borane,2-Picoline Borane, 2,6-diborane-methanol, dimethylamine-borane,t-BuMe^(i)PrN—BH₃, benzylamine-BH₃ or 5-ethyl-2-methylpyridine borane(PEMB). In a preferred embodiment, the reducing agent is sodiumcyanoborohydride.

In a preferred embodiment, the duration of step (b) is between 1 and 50,5 and 30, 10 and 30, 15 and 30, 20 and 30, 5 and 25, 10 and 25, 15 and25 hours. In a preferred embodiment, the duration of step (b) is about20, 21, 22, 23, 24, 25, 26, 27, 28 hours.

In a preferred embodiment, the temperature of the reaction in step (b)is maintained between 10 and 40° C., 15 and 30° C., 20 and 26° C., 21and 25° C. In a preferred embodiment, the temperature of the reaction instep (b) is maintained at about 21, 22, 23, 24 or 25° C.

In a preferred embodiment, the process for the preparation of animmunogenic conjugate comprising serotype 10A, 22F or 33F polysaccharidecovalently linked to a carrier protein further comprises a step (step c)of capping unreacted aldehyde (quenching) by addition of NaBH₄.

In a preferred embodiment, in step (c), the unreacted aldehydes arecapped by the addition of 0.1 to 10 molar equivalents, 0.5 to 5 molarequivalents or 1 to 3 molar equivalents of NaBH₄. In a preferredembodiment, in step (c), the unreacted aldehydes are capped by theaddition of about 2 molar equivalents of NaBH₄.

In a preferred embodiment, the duration of step (c) is between 0.1 and10, 0.5 and 5 or 2 and 4 hours. In a preferred embodiment, the durationof step (c) is about 3 hours.

In a preferred embodiment, the temperature of the reaction in step (c)is maintained between 10 to 40° C., 15 to 30° C. or 20 to 26° C. In apreferred embodiment, the temperature of the reaction in step (c) ismaintained at about 23° C.

After conjugation of serotype 10A, 22F or 33F polysaccharide to thecarrier protein, the polysaccharide-protein conjugate can be purified(enriched with respect to the amount of polysaccharide-proteinconjugate) by a variety of techniques known to the skilled person. Thesetechniques include dialysis, concentration/diafiltration operations,tangential flow filtration precipitation/elution, column chromatography(DEAE or hydrophobic interaction chromatography), and depth filtration.

In a preferred embodiment the carrier protein is non-toxic andnon-reactogenic and obtainable in sufficient amount and purity. Carrierproteins should be amenable to standard conjugation procedures.

In a preferred embodiment, the activated serotype 10A, 22F or 33Fpolysaccharide is conjugated to a carrier protein which is selected inthe group consisting of: DT (Diphtheria toxin), TT (tetanus toxid) orfragment C of TT, CRM197 (a nontoxic but antigenically identical variantof diphtheria toxin) other DT point mutants, such as CRM176, CRM228, CRM45 (Uchida et al J. Biol. Chem. 218; 3838-3844, 1973); CRM 9, CRM102,CRM 103 and CRM107 and other mutations described by Nicholls and Youlein Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992;deletion or mutation of Glu-148 to Asp, Gln or Ser and/or Ala 158 to Glyand other mutations disclosed in U.S. Pat. No. 4,709,017 or 4,950,740;mutation of at least one or more residues Lys 516, Lys 526, Phe 530and/or Lys 534 and other mutations disclosed in U.S. Pat. No. 5,917,017or 6,455,673; or fragment disclosed in U.S. Pat. No. 5,843,711,pneumococcal pneumolysin (Kuo et al (1995) Infect Immun 63; 2706-13)including ply detoxified in some fashion for example dPLY-GMBS (WO04081515, PCT/EP2005/010258) or dPLY-formol, PhtX, including PhtA, PhtB,PhtD, PhtE (sequences of PhtA, PhtB, PhtD or PhtE are disclosed in WO00/37105 or WO 00/39299) and fusions of Pht proteins for example PhtDEfusions, PhtBE fusions, Pht A-E (WO 01/98334, WO 03/54007,WO2009/000826), OMPC (meningococcal outer membrane protein—usuallyextracted from N. meningitidis serogroup B—EP0372501), PorB (from N.meningitidis), PD (Haemophilus influenza protein D—see, e.g., EP 0 594610 B), or immunologically functional equivalents thereof, syntheticpeptides (EP0378881, EP0427347), heat shock proteins (WO 93/17712, WO94/03208), pertussis proteins (WO 98/58668, EP0471 177), cytokines,lymphokines, growth factors or hormones (WO10 91/01146), artificialproteins comprising multiple human CD4+ T cell epitopes from variouspathogen derived antigens (Falugi et al (2001) Eur J Immunol 31;3816-3824) such as N19 protein (Baraldoi et al (2004) Infect Immun 72;4884-7) pneumococcal surface protein PspA (WO 02/091998), iron uptakeproteins (WO 01/72337), toxin A or B of C. difficile (WO 00/61761). Inan embodiment, the activated serotype 10A, 22F or 33F polysaccharide isconjugated to DT (Diphtheria toxoid). In another embodiment, theactivated serotype 10A, 22F or 33F polysaccharide is conjugated to TT(tetanus toxid). In another embodiment, the activated serotype 10A, 22For 33F polysaccharide is conjugated to fragment C of TT. In anotherembodiment, the activated serotype 10A, 22F or 33F polysaccharide isconjugated to PD (Haemophilus influenza protein D—see, e.g., EP 0 594610 B).

In a preferred embodiment, the activated serotype 10A, 22F or 33Fpolysaccharide is conjugated to CRM₁₉₇ protein. The CRM₁₉₇ protein is anontoxic form of diphtheria toxin but is immunologicallyindistinguishable from the diphtheria toxin. CRM₁₉₇ is produced by C.diphtheriae infected by the nontoxigenic phage β197^(tox−) created bynitrosoguanidine mutagenesis of the toxigenic corynephage beta (Uchida,T. et al. 1971, Nature New Biology 233:8-11). CRM₁₉₇ is purified throughultrafiltration, ammonium sulfate precipitation, and ion-exchangechromatography. The CRM₁₉₇ protein has the same molecular weight as thediphtheria toxin but differs therefrom by a single base change (guanineto adenine) in the structural gene. This single base change causes anamino acid substitution glutamic acid for glycine) in the mature proteinand eliminates the toxic properties of diphtheria toxin. The CRM₁₉₇protein is a safe and effective T-cell dependent carrier forsaccharides. Further details about CMR₁₉₇ and production thereof can befound e.g. in U.S. Pat. No. 5,614,382.

In a preferred embodiment, the activated serotype 10A, 22F or 33Fpolysaccharide disclosed herein is conjugated to CRM₁₉₇ by a processcomprising the step of:

(a) compounding the activated serotype 10A, 22F or 33F polysaccharidewith CRM₁₉₇;(b) reacting the compounded activated serotype 10A, 22F or 33Fpolysaccharide and CRM₁₉₇ with sodium cyanoborohydride to form aserotype 10A, 22F or 33F polysaccharide:CRM197 conjugate.

In a preferred embodiment, the activated serotype 10A, 22F or 33Fpolysaccharide disclosed herein is conjugated to CRM197 by a processcomprising the step of:

(a) compounding the activated serotype 10A, 22F or 33F polysaccharidewith a CRM₁₉₇;(b) reacting the compounded activated serotype 10A, 22F or 33Fpolysaccharide and CRM₁₉₇ with sodium cyanoborohydride to form aserotype 10A, 22F or 33F polysaccharide:CRM197 conjugate;wherein steps (a) and (b) are carried out in DMSO.

In a preferred embodiment, the activated serotype 10A, 22F or 33Fpolysaccharide disclosed herein is conjugated to CRM₁₉₇ by a processcomprising the step of:

(a) compounding the activated serotype 10A, 22F or 33F polysaccharidewith a CRM₁₉₇;(b) reacting the compounded activated serotype 10A, 22F or 33Fpolysaccharide and CRM₁₉₇ with 0.5 to 2 molar equivalent sodiumcyanoborohydride to form a serotype 10A, 22F or 33Fpolysaccharide:CRM₁₉₇ conjugate;wherein steps (a) and (b) are carried out in DMSO.

In an embodiment, the invention relates to an immunogenic conjugateobtained or a obtainable by a process disclosed herein. In a preferredembodiment, the invention relates to an immunogenic conjugate obtainedor obtainable by conjugating a serotype 10A, 22F or 33F activatedpolysaccharide as disclosed herein to a carrier protein by reductiveamination. In a preferred embodiment, the invention relates to animmunogenic conjugate obtained or obtainable by conjugating a serotype10A, 22F or 33F activated polysaccharide as disclosed herein to acarrier protein by reductive amination in DMSO. In a preferredembodiment, the carrier protein is CRM₁₉₇.

Serotype 10A Immunogenic Conjugate

In a preferred embodiment, the serotype 10A immunogenic conjugate has amolecular weight between 500 and 15000; 500 and 10000; 2000 and 10000;or 3000 and 8000 kDa. In a preferred embodiment, the serotype 10Aimmunogenic conjugate has a molecular weight between 3000 and 8000 kDa.The molecular weight of the immunogenic conjugate is measured bySEC-MALLS.

In a preferred embodiment, the serotype 10A immunogenic conjugatecomprises less than about 50, 45, 40, 35, 30, 25, 20 or 15% of freeserotype 10A polysaccharide compared to the total amount of serotype 10Apolysaccharide. In a preferred embodiment the serotype 10A immunogenicconjugate comprises less than about 25% of free serotype 10Apolysaccharide compared to the total amount of serotype 10Apolysaccharide. In a preferred embodiment the serotype 10A immunogenicconjugate comprises less than about 20% of free serotype 10Apolysaccharide compared to the total amount of serotype 10Apolysaccharide. In a preferred embodiment the serotype 10A immunogenicconjugate comprises less than about 15% of free serotype 10Apolysaccharide compared to the total amount of serotype 10Apolysaccharide.

In a preferred embodiment, the ratio (weight by weight) of serotype 10Apolysaccharide to carrier protein in the conjugate is between 0.5 and 3.In a preferred embodiment, the ratio of serotype 10A polysaccharide tocarrier protein in the conjugate is between 0.5 and 2, 0.5 and 1.5, 0.5and 1, 1 and 1.5, 1 and 2. In a preferred embodiment, the ratio ofserotype 10A polysaccharide to carrier protein in the conjugate isbetween 0.8 and 1.4. In a preferred embodiment, the ratio of serotype10A capsular polysaccharide to carrier protein in the conjugate isbetween 0.8 and 1.2.

Size exclusion chromatography media (CL-4B) can be used to determine therelative molecular size distribution of the conjugate. Size ExclusionChromatography (SEC) is used in gravity fed columns to profile themolecular size distribution of conjugates. Large molecules excluded fromthe pores in the media elute more quickly than small molecules. Fractioncollectors are used to collect the column eluate. The fractions aretested colorimetrically by saccharide assay. For the determination ofKd, columns are calibrated to establish the fraction at which moleculesare fully excluded (V₀), (Kd=0), and the fraction representing themaximum retention (V_(i)), (Kd=1). The fraction at which a specifiedsample attribute is reached (V_(e)), is related to Kd by the expression,Kd=(V_(e)−V₀)/(V_(i)−V₀).

In a preferred embodiment, at least 30% of the serotype 10A immunogenicconjugate has a Kd below or equal to 0.3 in a CL-4B column. In apreferred embodiment, at least 40% of the immunogenic conjugate has a Kdbelow or equal to 0.3 in a CL-4B column. In a preferred embodiment, atleast 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the serotype 10Aimmunogenic conjugate has a Kd below or equal to 0.3 in a CL-4B column.In a preferred embodiment, at least 60% of the serotype 10A immunogenicconjugate has a Kd below or equal to 0.3 in a CL-4B column. In apreferred embodiment, between 50% and 80% of the serotype 10Aimmunogenic conjugate has a Kd below or equal to 0.3 in a CL-4B column.

The degree of conjugation is the number of lysine residues in thecarrier protein that are conjugated to the polysaccharide of interest.The evidence for lysine modification of the carrier protein, due tocovalent linkages to the polysaccharides, is obtained by amino acidanalysis using routine methods known to those of skill in the art.Conjugation results in a reduction in the number of lysine residuesrecovered, compared to the CRM₁₉₇ protein starting material used togenerate the conjugate materials.

In a preferred embodiment, the degree of conjugation of the immunogenicconjugate is between 2 and 15, 2 and 13, 2 and 10, 2 and 8, 2 and 6, 2and 5, 2 and 4, 3 and 15, 3 and 13, 3 and 10, 3 and 8, 3 and 6, 3 and 5,3 and 4, 5 and 15, 5 an 10, 8 and 15, 8 and 12, 10 and 15 or 10 and 12.In a preferred embodiment, the degree of conjugation of the immunogenicconjugate is between 6 and 8.

Serotype 22F Immunogenic Conjugate

In a preferred embodiment, the immunogenic conjugate comprises at least0.1, 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7 or about 0.8 mM acetate per mMserotype 22F polysaccharide. In a preferred embodiment, the immunogenicconjugate comprises at least 0.5, 0.6 or 0.7 mM acetate per mM serotype22F polysaccharide. In a preferred embodiment, the immunogenic conjugatecomprises at least 0.6 mM acetate per mM serotype 22F polysaccharide. Ina preferred embodiment, the immunogenic conjugate comprises at least 0.7mM acetate per mM serotype 22F polysaccharide.

In a preferred embodiment, the ratio of mM acetate per mM serotype 22Fpolysaccharide in the immunogenic conjugate to mM acetate per mMserotype 22F polysaccharide in the isolated polysaccharide is at least0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or 0.95. In a preferredembodiment, the ratio of mM acetate per mM serotype 22F polysaccharidein the immunogenic conjugate to mM acetate per mM serotype 22Fpolysaccharide in the isolated polysaccharide is at least 0.7. In apreferred embodiment, the ratio of mM acetate per mM serotype 22Fpolysaccharide in the immunogenic conjugate to mM acetate per mMserotype 22F polysaccharide in the isolated polysaccharide is at least0.9.

In a preferred embodiment, the ratio of mM acetate per mM serotype 22Fpolysaccharide in the immunogenic conjugate to mM acetate per mMserotype 22F polysaccharide in the activated polysaccharide is at least0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or 0.95. In a preferredembodiment, the ratio of mM acetate per mM serotype 22F polysaccharidein the immunogenic conjugate to mM acetate per mM serotype 22Fpolysaccharide in the activated polysaccharide is at least 0.7. In apreferred embodiment, the ratio of mM acetate per mM serotype 22Fpolysaccharide in the immunogenic conjugate to mM acetate per mMserotype 22F polysaccharide in the activated polysaccharide is at least0.9.

In a preferred embodiment, the serotype 22F immunogenic conjugate has amolecular weight between 400 and 15000; 500 and 10000; 2000 and 10000kDa; 3000 and 8000 kDa; or 3000 and 5000 kDa. In a preferred embodiment,the serotype 22F immunogenic conjugate has a molecular weight between3000 and 5000 kDa. The molecular weight of the immunogenic conjugate ismeasured by SEC-MALLS.

In a preferred embodiment, the serotype 22F immunogenic conjugatecomprises less than about 50, 45, 40, 35, 30, 25, 20 or 15% of freeserotype 22F polysaccharide compared to the total amount of serotype 22Fpolysaccharide. In a preferred embodiment the serotype 22F immunogenicconjugate comprises less than about 40% of free serotype 22Fpolysaccharide compared to the total amount of serotype 22Fpolysaccharide. In a preferred embodiment the serotype 22F immunogenicconjugate comprises less than about 25% of free serotype 22Fpolysaccharide compared to the total amount of serotype 22Fpolysaccharide. In a preferred embodiment the serotype 22F immunogenicconjugate comprises less than about 20% of free serotype 22Fpolysaccharide compared to the total amount of serotype 22Fpolysaccharide. In a preferred embodiment the serotype 22F immunogenicconjugate comprises less than about 15% of free serotype 22Fpolysaccharide compared to the total amount of serotype 22Fpolysaccharide.

In a preferred embodiment, the ratio (weight by weight) of serotype 22Fpolysaccharide to carrier protein in the conjugate is between 0.5 and 3.In a preferred embodiment, the ratio of serotype 22F polysaccharide tocarrier protein in the conjugate is between 0.5 and 2, 0.5 and 1.5, 0.8and 1.2, 0.5 and 1, 1 and 1.5, or 1 and 2. In a preferred embodiment,the ratio of serotype 22F polysaccharide to carrier protein in theconjugate is between 0.8 and 1.2. In a preferred embodiment, the ratioof serotype 22F capsular polysaccharide to carrier protein in theconjugate is between 0.9 and 1.1.

In a preferred embodiment, at least 30% of the serotype 22F immunogenicconjugate has a Kd below or equal to 0.3 in a CL-4B column. In apreferred embodiment, at least 40% of the immunogenic conjugate has a Kdbelow or equal to 0.3 in a CL-4B column. In a preferred embodiment, atleast 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the serotype 22Fimmunogenic conjugate has a Kd below or equal to 0.3 in a CL-4B column.In a preferred embodiment, at least 60% of the serotype 22F immunogenicconjugate has a Kd below or equal to 0.3 in a CL-4B column. In apreferred embodiment, between 50% and 80% of the serotype 22Fimmunogenic conjugate has a Kd below or equal to 0.3 in a CL-4B column.In a preferred embodiment, between 65% and 80% of the serotype 22Fimmunogenic conjugate has a Kd below or equal to 0.3 in a CL-4B column.

In a preferred embodiment, the degree of conjugation of the immunogenicconjugate is between 2 and 15, 2 and 13, 2 and 10, 2 and 8, 2 and 6, 2and 5, 2 and 4, 3 and 15, 3 and 13, 3 and 10, 3 and 8, 3 and 6, 3 and 5,3 and 4, 4 and 7, 5 and 15, 5 an 10, 8 and 15, 8 and 12, 10 and 15 or 10and 12. In a preferred embodiment, the degree of conjugation of theimmunogenic conjugate is between 4 and 7.

Serotype 33F Immunogenic Conjugate

In a preferred embodiment, the immunogenic conjugate comprises at least0.1, 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7 or about 0.8 mM acetate per mMserotype 33F polysaccharide. In a preferred embodiment, the immunogenicconjugate comprises at least 0.5, 0.6 or 0.7 mM acetate per mM serotype33F polysaccharide. In a preferred embodiment, the immunogenic conjugatecomprises at least 0.6 mM acetate per mM serotype 33F polysaccharide. Ina preferred embodiment, the immunogenic conjugate comprises at least 0.7mM acetate per mM serotype 33F polysaccharide.

In a preferred embodiment, the ratio of mM acetate per mM serotype 33Fpolysaccharide in the immunogenic conjugate to mM acetate per mMserotype 33F polysaccharide in the isolated polysaccharide is at least0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or 0.95. In a preferredembodiment, the ratio of mM acetate per mM serotype 33F polysaccharidein the immunogenic conjugate to mM acetate per mM serotype 33Fpolysaccharide in the isolated polysaccharide is at least 0.7. In apreferred embodiment, the ratio of mM acetate per mM serotype 33Fpolysaccharide in the immunogenic conjugate to mM acetate per mMserotype 33F polysaccharide in the isolated polysaccharide is at least0.9.

In a preferred embodiment, the ratio of mM acetate per mM serotype 33Fpolysaccharide in the immunogenic conjugate to mM acetate per mMserotype 33F polysaccharide in the activated polysaccharide is at least0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or 0.95. In a preferredembodiment, the ratio of mM acetate per mM serotype 33F polysaccharidein the immunogenic conjugate to mM acetate per mM serotype 33Fpolysaccharide in the activated polysaccharide is at least 0.7. In apreferred embodiment, the ratio of mM acetate per mM serotype 33Fpolysaccharide in the immunogenic conjugate to mM acetate per mMserotype 33F polysaccharide in the activated polysaccharide is at least0.9.

In a preferred embodiment, the serotype 33F immunogenic conjugate has amolecular weight between 500 and 30000; 500 and 25000; 500 and 20000;500 and 15000; 500 and 10000; 1000 and 10000; 1000 and 8000; 1000 and5000; 2000 and 10000 kDa; 2000 and 8000; or 2000 and 5000 kDa. In apreferred embodiment, the serotype 33F immunogenic conjugate has amolecular weight between 1000 and 5000 kDa. The molecular weight of theimmunogenic conjugate is measured by SEC-MALLS.

In a preferred embodiment, the serotype 33F immunogenic conjugatecomprises less than about 50, 45, 40, 35, 30, 25, 20 or 15% of freeserotype 33F polysaccharide compared to the total amount of serotype 33Fpolysaccharide. In a preferred embodiment the serotype 33F immunogenicconjugate comprises less than about 25% of free serotype 33Fpolysaccharide compared to the total amount of serotype 33Fpolysaccharide. In a preferred embodiment the serotype 33F immunogenicconjugate comprises less than about 20% of free serotype 33Fpolysaccharide compared to the total amount of serotype 33Fpolysaccharide. In a preferred embodiment the serotype 33F immunogenicconjugate comprises less than about 15% of free serotype 33Fpolysaccharide compared to the total amount of serotype 33Fpolysaccharide.

In a preferred embodiment, the ratio (weight by weight) of serotype 33Fpolysaccharide to carrier protein in the conjugate is between 0.4 and 3.In a preferred embodiment, the ratio of serotype 33F polysaccharide tocarrier protein in the conjugate is between 0.5 and 2, 0.5 and 1.5, 0.5and 1, 1 and 1.5, 1 and 2. In a preferred embodiment, the ratio ofserotype 33F polysaccharide to carrier protein in the conjugate isbetween 0.5 and 1.5. In a preferred embodiment, the ratio of serotype33F capsular polysaccharide to carrier protein in the conjugate isbetween 0.5 and 1.2.

In a preferred embodiment, at least 30% of the serotype 33F immunogenicconjugate has a Kd below or equal to 0.3 in a CL-4B column. In apreferred embodiment, at least 40% of the immunogenic conjugate has a Kdbelow or equal to 0.3 in a CL-4B column. In a preferred embodiment, atleast 45%, 50%, 55%, 60%, 65% of the serotype 33F immunogenic conjugatehas a Kd below or equal to 0.3 in a CL-4B column. In a preferredembodiment, at least 60% of the serotype 33F immunogenic conjugate has aKd below or equal to 0.3 in a CL-4B column. In a preferred embodiment,between 40% and 80% of the serotype 33F immunogenic conjugate has a Kdbelow or equal to 0.3 in a CL-4B column. In a preferred embodiment,between 45% and 65% of the serotype 33F immunogenic conjugate has a Kdbelow or equal to 0.3 in a CL-4B column.

In a preferred embodiment, the degree of conjugation of the immunogenicconjugate is between 1 and 15, 2 and 13, 2 and 10, 2 and 8, 2 and 6, 2and 5, 2 and 4, 3 and 15, 3 and 13, 3 and 10, 3 and 8, 3 and 6, 3 and 5,3 and 4, 5 and 15, 5 an 10, 8 and 15, 8 and 12, 10 and 15 or 10 and 12.In a preferred embodiment, the degree of conjugation of the immunogenicconjugate is between 3 and 6.

Immunogenic Composition

The term “immunogenic composition” relates to any pharmaceuticalcomposition containing an antigen, e.g., a microorganism or a componentthereof, which composition can be used to elicit an immune response in asubject.

As used herein, “immunogenic” means an ability of an antigen (or anepitope of the antigen), such as a bacterial capsular polysaccharide, ora glycoconjugate or immunogenic composition comprising an antigen, toelicit an immune response in a host such as a mammal, either humorallyor cellularly mediated, or both.

In a preferred embodiment, the immunogenic composition comprises aserotype 10A, 22F or 33F conjugate obtained by a process disclosedherein. In a preferred embodiment, the immunogenic compositioncomprising a serotype 10A, 22F or 33F conjugate obtainable by a processdisclosed herein.

In an embodiment, the immunogenic composition disclosed herein, whenadministered to a subject, induces the formation of antibodies capableof binding to serotype 10A Streptococcus pneumonia. In an embodiment,the immunogenic composition disclosed herein, when administered to asubject, induces the formation of antibodies capable of binding toserotype 10A Streptococcus pneumonia as measured by a standard ELISAassay.

In an embodiment, the immunogenic composition disclosed herein, whenadministered to a subject, induces the formation of antibodies capableof killing serotype 10A Streptococcus pneumonia in an opsonophagocytosisassay as disclosed herein.

In an embodiment, the immunogenic composition disclosed herein, whenadministered to a subject, induces the formation of antibodies capableof binding to serotype 22F Streptococcus pneumonia. In an embodiment,the immunogenic composition disclosed herein, when administered to asubject, induces the formation of antibodies capable of binding toserotype 22F Streptococcus pneumonia as measured by a standard ELISAassay.

In an embodiment, the immunogenic composition disclosed herein, whenadministered to a subject, induces the formation of antibodies capableof killing serotype 22F Streptococcus pneumonia in an opsonophagocytosisassay as disclosed herein.

In an embodiment, the immunogenic composition disclosed herein, whenadministered to a subject, induces the formation of antibodies capableof binding to serotype 33F Streptococcus pneumonia. In an embodiment,the immunogenic composition disclosed herein, when administered to asubject, induces the formation of antibodies capable of binding toserotype 33F Streptococcus pneumonia as measured by a standard ELISAassay.

In an embodiment, the immunogenic composition disclosed herein, whenadministered to a subject, induces the formation of antibodies capableof killing serotype 33F Streptococcus pneumonia in an opsonophagocytosisassay as disclosed herein.

Formulation of the immunogenic composition of the present invention canbe accomplished using art-recognized methods. For instance, the serotype10A, 22F or 33F conjugates can be formulated with a physiologicallyacceptable vehicle to prepare the composition. Examples of such vehiclesinclude, but are not limited to, water, buffered saline, polyols (e.g.,glycerol, propylene glycol, liquid polyethylene glycol) and dextrosesolutions.

In a preferred embodiment, the immunogenic composition may comprise atleast one additional antigen. In a preferred embodiment, the immunogeniccomposition may comprises at least one additional Streptococcuspneumoniae capsular polysaccharide.

In a preferred embodiment, the immunogenic composition may comprise atleast one additional Streptococcus pneumoniae capsular polysaccharideconjugated to a carrier protein. In a preferred embodiment, said carrierprotein is CRM₁₉₇.

In certain embodiments, the immunogenic composition comprises one ormore adjuvants. As defined herein, an “adjuvant” is a substance thatserves to enhance the immunogenicity of an immunogenic composition ofthis invention. Thus, adjuvants are often given to boost the immuneresponse and are well known to the skilled artisan. Suitable adjuvantsto enhance effectiveness of the composition include, but are not limitedto:

(1) aluminum salts (alum), such as aluminum hydroxide, aluminumphosphate, aluminum sulfate, etc.;(2) oil-in-water emulsion formulations (with or without other specificimmunostimulating agents such as muramyl peptides (defined below) orbacterial cell wall components), such as, for example,(a) MF59 (PCT Pub. No. WO 90/14837), containing 5% Squalene, 0.5%5 Tween80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE(see below, although not required)) formulated into submicron particlesusing a microfluidizer such as Model 11OY microfluidizer (Microfluidics,Newton, Mass.),(b) SAF, containing 10% Squalene, 0.4% Tween 80, 5% pluronic-blockedpolymer L121, and thr-MDP (see below) either microfluidized into asubmicron emulsion or vortexed to generate a larger particle sizeemulsion, and(c) Ribi™ adjuvant system (RAS), (Corixa, Hamilton, Mont.) containing 2%Squalene, 0.2% Tween 80, and one or more bacterial cell wall componentsfrom the group consisting of 3-O-deaylated monophosphorylipid A (MPL™)described in U.S. Pat. No. 4,912,094 (Corixa), trehalose dimycolate(TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™);(3) saponin adjuvants, such as Quil A or STIMULON™ QS-21 (Antigenics,Framingham, Mass.) (U.S. Pat. No. 5,057,540) may be used or particlesgenerated therefrom such as ISCOMs (immunostimulating complexes);(4) bacteriallipopolysaccharides, synthetic lipid A analogs such asaminoalkyl glucosamine phosphate compounds (AGP), or derivatives oranalogs thereof, which are available from Corixa, and which aredescribed in U.S. Pat. No. 6,113,918; one such AGP is2-[(R)-3-Tetradecanoyloxytetradecanoylamino]ethyl2-Deoxy-4-Ophosphono-3-O—[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3tetradecanoyloxytetradecanoylamino]-b-D-glucopyranoside,which is also know as 529 (formerly known as RC529), which is formulatedas an aqueous form or as a stable emulsion, synthetic polynucleotidessuch as oligonucleotides containing CpG motif(s) (U.S. Pat. No.6,207,646);(5) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6,IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g., gamma interferon),granulocyte macrophage colony stimulating factor (GM-CSF), macrophagecolony stimulating factor (M-CSF), tumor necrosis factor (TNF),costimulatory molecules 87-1 and 87-2, etc.;(6) detoxified mutants of a bacterial ADP-ribosylating toxin such as acholera toxin (CT) either in a wild-type or mutant form, for example,where the glutamic acid at amino acid position 29 is replaced by anotheramino acid, preferably a histidine, in accordance with publishedinternational patent application number WO 00/18434 (see also WO02/098368 and WO 02/098369), a pertussis toxin (PT), or an E. coliheat-labile toxin (LT), particularly LT-K63, LT-R72, CT-S109, PT-K9/G129(see, e.g., WO 93/13302 and WO 92/19265); and(7) other substances that act as immunostimulating agents to enhance theeffectiveness of the composition.

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

In an embodiment of the present invention, the immunogenic compositionsas disclosed herein comprise a CpG Oligonucleotide as adjuvant. A CpGoligonucleotide as used herein refers to an immunostimulatory CpGoligodeoxynucleotide (CpG ODN), and accordingly these terms are usedinterchangeably unless otherwise indicated. Immunostimulatory CpGoligodeoxynucleotides contain one or more immunostimulatory CpG motifsthat are unmethylated cytosine-guanine dinucleotides, optionally withincertain preferred base contexts. The methylation status of the CpGimmunostimulatory motif generally refers to the cytosine residue in thedinucleotide. An immunostimulatory oligonucleotide containing at leastone unmethylated CpG dinucleotide is an oligonucleotide which contains a5′ unmethylated cytosine linked by a phosphate bond to a 3′ guanine, andwhich activates the immune system through binding to Toll-like receptor9 (TLR-9). In another embodiment the immunostimulatory oligonucleotidemay contain one or more methylated CpG dinucleotides, which willactivate the immune system through TLR9 but not as strongly as if theCpG motif(s) was/were unmethylated. CpG immunostimulatoryoligonucleotides may comprise one or more palindromes that in turn mayencompass the CpG dinucleotide. CpG oligonucleotides have been describedin a number of issued patents, published patent applications, and otherpublications, including U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806;6,218,371; 6,239,116; and 6,339,068.

In an embodiment of the present invention, the immunogenic compositionsas disclosed herein comprise any of the CpG Oligonucleotide described atpages 3 lines 22 to page 12 line 36 of WO2010/125480.

Different classes of CpG immunostimulatory oligonucleotides have beenidentified. These are referred to as A, B, C and P class, and aredescribed in greater detail at pages 3 lines 22 to page 12 line 36 ofWO2010/125480. Methods of the invention embrace the use of thesedifferent classes of CpG immunostimulatory oligonucleotides.

In an embodiment of the present invention, the immunogenic compositionsas disclosed herein comprise an A class CpG Oligonucleotide. Preferably,the “A class” CpG oligonucleotide of the invention has the followingnucleic acid sequence: 5′ GGGGACGACGTCGTGGGGGGG 3′ (SEQ ID NO: 1). Somenon-limiting examples of A-Class oligonucleotides include: 5′G*G*G_G_A_C_G_A_C_G_T_C_G_T_G_G*G*G*G*G*G 3′ (SEQ ID NO: 2); wherein *refers to a phosphorothioate bond and _ refers to a phosphodiester bond.

In an embodiment of the present invention, the immunogenic compositionsas disclosed herein comprise a B class CpG Oligonucleotide. In oneembodiment, the CpG oligonucleotide for use in the present invention isa B class CpG oligonucleotide represented by at least the formula:

5′ X₁X₂CGX₃X₄ 3′, wherein X₁, X₂, X₃, and X₄ are nucleotides. In oneembodiment, X₂ is adenine, guanine, or thymine. In another embodiment,X₃ is cytosine, adenine, or thymine.

The B class CpG oligonucleotide sequences of the invention are thosebroadly described above in U.S. Pat. Nos. 6,194,388, 6,207,646,6,214,806, 6,218,371, 6,239,116 and 6,339,068. Exemplary sequencesinclude but are not limited to those disclosed in these latterapplications and patents.

In an embodiment, the “B class” CpG oligonucleotide of the invention hasthe following nucleic acid sequence:

(SEQ ID NO: 3) 5′ TCGTCGTTTTTCGGTGCTTTT 3′,  or (SEQ ID NO: 4)5′ TCGTCGTTTTTCGGTCGTTTT 3′,  or (SEQ ID NO: 5)5′ TCGTCGTTTTGTCGTTTTGTCGTT 3′,  or (SEQ ID NO: 6)5′ TCGTCGTTTCGTCGTTTTGTCGTT 3′,  or (SEQ ID NO: 7)5′ TCGTCGTTTTGTCGTTTTTTTCGA 3′.

In any of these sequences, all of the linkages may be allphosphorothioate bonds. In another embodiment, in any of thesesequences, one or more of the linkages may be phosphodiester, preferablybetween the “C” and the “G” of the CpG motif making a semi-soft CpGoligonucleotide. In any of these sequences, an ethyl-uridine or ahalogen may substitute for the 5′ T; examples of halogen substitutionsinclude but are not limited to bromo-uridine or iodo-uridinesubstitutions.

Some non-limiting examples of B-Class oligonucleotides include:

(SEQ ID NO: 8) 5′ T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*G* C*T*T*T*T 3′, or(SEQ ID NO: 9) 5′ T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*C* G*T*T*T*T 3′, or(SEQ ID NO: 10) 5′ T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*  T*T*G*T*C*G*T*T 3′,or (SEQ ID NO: 11) 5′ T*C*G*T*C*G*T*T*T*C*G*T*C*G*T*T* T*T*G*T*C*G*T*T 3′, or (SEQ ID NO: 12)5′ T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T* T*T*T*T*T*C*G*A 3′.wherein * refers to a phosphorothioate bond.

In an embodiment of the present invention, the immunogenic compositionsas disclosed herein comprise a C class CpG Oligonucleotide. In anembodiment, the “C class” CpG oligonucleotides of the invention has thefollowing nucleic acid sequence:

(SEQ ID NO: 13) 5′ TCGCGTCGTTCGGCGCGCGCCG 3′, or (SEQ ID NO: 14)5′ TCGTCGACGTTCGGCGCGCGCCG 3′,  or (SEQ ID NO: 15)5′ TCGGACGTTCGGCGCGCGCCG 3′,  or (SEQ ID NO: 16)5′ TCGGACGTTCGGCGCGCCG 3′,  or (SEQ ID NO: 17)5′ TCGCGTCGTTCGGCGCGCCG 3′, or (SEQ ID NO: 18)5′ TCGACGTTCGGCGCGCGCCG 3′,  or (SEQ ID NO: 19)5′ TCGACGTTCGGCGCGCCG 3′,  or (SEQ ID NO: 20) 5′ TCGCGTCGTTCGGCGCCG 3′, or (SEQ ID NO: 21) 5′ TCGCGACGTTCGGCGCGCGCCG 3′,  or (SEQ ID NO: 22)5′ TCGTCGTTTTCGGCGCGCGCCG 3′,  or (SEQ ID NO: 23)5′ TCGTCGTTTTCGGCGGCCGCCG 3′,  or (SEQ ID NO: 24)5′ TCGTCGTTTTACGGCGCCGTGCCG 3′,  or (SEQ ID NO: 25)5′ TCGTCGTTTTCGGCGCGCGCCGT 3′.

In any of these sequences, all of the linkages may be allphosphorothioate bonds. In another embodiment, in any of thesesequences, one or more of the linkages may be phosphodiester, preferablybetween the “C” and the “G” of the CpG motif making a semi-soft CpGoligonucleotide.

Some non-limiting examples of C-Class oligonucleotides include:

(SEQ ID NO: 26) 5′ T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C_G* C*G*C*C*G 3′, or(SEQ ID NO: 27) 5′ T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_ G*C*G*C*C*G 3′, or(SEQ ID NO: 28) 5′ T*C_G*G*A*C_G*T*T*C_G*G*C*G*C_G*C*  G*C*C*G 3′, or(SEQ ID NO: 29) 5′ T*C_G*G*A*C_G*T*T*C_G*G*C*G*C*G*C* C*G 3′, or(SEQ ID NO: 30) 5′ T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*G* C*C*G 3′, or(SEQ ID NO: 31) 5′ T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G* C*C*G 3′, or(SEQ ID NO: 32) 5′ T*C_G*A*C_G*T*T*C_G*G*C*G*C*G*C*C* G 3′, or(SEQ ID NO: 33) 5′ T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*C* G 3′, or(SEQ ID NO: 34) 5′ T*C_G*C_G*A*C_G*T*T*C_G*G*C*G*C_G* C*G*C*C*G 3′, or(SEQ ID NO: 35) 5′ T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*C*G* C*G*C*C*G 3′, or(SEQ ID NO: 36) 5′ T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*G*C* C*G*C*C*G 3′, or(SEQ ID NO: 37) 5′ T*C*G*T*C_G*T*T*T*T*A*C_G*G*C*G*C* C_G*T*G*C*C*G 3′,or (SEQ ID NO: 38) 5′ T*C_G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*  C*G*C*C*G*T 3′wherein * refers to a phosphorothioate bond and _ refers to aphosphodiester bond.

In any of these sequences, an ethyl-uridine or a halogen may substitutefor the 5′ T; examples of halogen substitutions include but are notlimited to bromo-uridine or iodo-uridine substitutions.

In an embodiment of the present invention, the immunogenic compositionsas disclosed herein comprise a P class CpG Oligonucleotide. In anembodiment, the CpG oligonucleotide for use in the present invention isa P class CpG oligonucleotide containing a 5′ TLR activation domain andat least two palindromic regions, one palindromic region being a 5′palindromic region of at least 6 nucleotides in length and connected toa 3′ palindromic region of at least 8 nucleotides in length eitherdirectly or through a spacer, wherein the oligonucleotide includes atleast one YpR dinucleotide. In an embodiment, said oligoonucleotide isnot T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G (SEQ ID NO: 27). Inone embodiment the a P class CpG oligonucleotide includes at least oneunmethylated CpG dinucleotide. In another embodiment the TLR activationdomain is TCG, TTCG, TTTCG, TYpR, TTYpR, TTTYpR, UCG, UUCG, UUUCG, TTT,or TTTT. In yet another embodiment the TLR activation domain is withinthe 5′ palindromic region. In another embodiment the TLR activationdomain is immediately 5′ to the 5′ palindromic region.

In an embodiment, the “P class” CpG oligonucleotides of the inventionhas the following nucleic acid sequence: 5′ TCGTCGACGATCGGCGCGCGCCG 3′(SEQ ID NO: 39).

In said sequences, all of the linkages may be all phosphorothioatebonds. In another embodiment, one or more of the linkages may bephosphodiester, preferably between the “C” and the “G” of the CpG motifmaking a semi-soft CpG oligonucleotide. In any of these sequences, anethyl-uridine or a halogen may substitute for the 5′ T; examples ofhalogen substitutions include but are not limited to bromo-uridine oriodo-uridine substitutions. A non-limiting example of P-Classoligonucleotides include:

5′ T*C_G*T*C_G*A*C_G*A*T*C_G*G*C*G*C_G*C*G*C*C*G 3′ (SEQ ID NO: 40)

wherein * refers to a phosphorothioate bond and _ refers to aphosphodiester bond.

In one embodiment the oligonucleotide includes at least onephosphorothioate linkage. In another embodiment all internucleotidelinkages of the oligonucleotide are phosphorothioate linkages. Inanother embodiment the oligonucleotide includes at least onephosphodiester-like linkage. In another embodiment thephosphodiester-like linkage is a phosphodiester linkage. In anotherembodiment a lipophilic group is conjugated to the oligonucleotide. Inone embodiment the lipophilic group is cholesterol.

In an embodiment, all the internucleotide linkage of the CpGoligonucleotides disclosed herein are phosphodiester bonds (“soft”oligonucleotides, as described in the PCT application WO2007/026190). Inanother embodiment, CpG oligonucleotides of the invention are renderedresistant to degradation (e.g., are stabilized). A “stabilizedoligonucleotide” refers to an oligonucleotide that is relativelyresistant to in vivo degradation (e.g. via an exo- or endo-nuclease).Nucleic acid stabilization can be accomplished via backbonemodifications. Oligonucleotides having phosphorothioate linkages providemaximal activity and protect the oligonucleotide from degradation byintracellular exo- and endo-nucleases.

The immunostimulatory oligonucleotides may have a chimeric backbone,which have combinations of phosphodiester and phosphorothioate linkages.For purposes of the instant invention, a chimeric backbone refers to apartially stabilized backbone, wherein at least one internucleotidelinkage is phosphodiester or phosphodiester-like, and wherein at leastone other internucleotide linkage is a stabilized internucleotidelinkage, wherein the at least one phosphodiester or phosphodiester-likelinkage and the at least one stabilized linkage are different. When thephosphodiester linkage is preferentially located within the CpG motifsuch molecules are called “semi-soft” as described in the PCTapplication WO2007/026190.

The size of the CpG oligonucleotide (i.e., the number of nucleotideresidues along the length of the oligonucleotide) also may contribute tothe stimulatory activity of the oligonucleotide. For facilitating uptakeinto cells, CpG oligonucleotide of the invention preferably have aminimum length of 6 nucleotide residues. Oligonucleotides of any sizegreater than 6 nucleotides (even many kb long) are capable of inducingan immune response if sufficient immunostimulatory motifs are present,because larger oligonucleotides are degraded inside cells. In certainembodiments, the CpG oligonucleotides are 6 to 100 nucleotides long,preferentially 8 to 30 nucleotides long. In important embodiments,nucleic acids and oligonucleotides of the invention are not plasm ids orexpression vectors.

In an embodiment, the CpG oligonucleotides disclosed herein comprisesubstitutions or modifications, such as in the bases and/or sugars asdescribed at paragraph 134 to 147 of WO2007/026190.

In an embodiment, the CpG oligonucleotide of the present invention ischemically modified. Examples of chemical modifications are known to theskilled person and are described, for example in Uhlmann E. et al.(1990), Chem. Rev. 90:543, S. Agrawal, Ed., Humana Press, Totowa, USA1993; Crooke, S. T. et al. (1996) Annu. Rev. Pharmacol. Toxicol.36:107-129; and Hunziker J. et al., (1995), Mod. Synth. Methods7:331-417. An oligonucleotide according to the invention may have one ormore modifications, wherein each modification is located at a particularphosphodiester internucleoside bridge and/or at a particular β-D-riboseunit and/or at a particular natural nucleoside base position incomparison to an oligonucleotide of the same sequence which is composedof natural DNA or RNA. In some embodiments of the invention,CpG-containing nucleic acids might be simply mixed with immunogeniccarriers according to methods known to those skilled in the art (see,e.g. WO03/024480).

In a particular embodiment of the present invention, any of theimmunogenic composition disclosed herein comprises from 2 μg to 100 mgof CpG oligonucleotide, preferably from 0.1 mg to 50 mg CpGoligonucleotide, preferably from 0.2 mg to 10 mg CpG oligonucleotide,preferably from 0.3 mg to 5 mg CpG oligonucleotide, even preferably from0.5 to 2 mg CpG oligonucleotide, even preferably from 0.75 to 1.5 mg CpGoligonucleotide. In a preferred embodiment, the immunogenic compositiondisclosed herein comprises approximately 1 mg CpG oligonucleotide.

In a preferred embodiment, the adjuvant is an aluminum-based adjuvantselected from the group consisting of aluminum phosphate, aluminumsulfate and aluminum hydroxide. In one embodiment, the immunogeniccompositions described herein comprise the adjuvant aluminum phosphate.

In a preferred embodiments, the immunogenic compositions of theinvention further comprise at least one of a buffer, a cryoprotectant, asalt, a divalent cation, a non-ionic detergent, an inhibitor of freeradical oxidation, a diluent or a carrier.

The immunogenic composition optionally can comprise a pharmaceuticallyacceptable carrier. Pharmaceutically acceptable carriers includecarriers approved by a regulatory agency of a Federal, a stategovernment, or other regulatory agency, or listed in the U.S.Pharmacopeia or other generally recognized pharmacopeia for use inanimals, including humans as well as non-human mammals. The term carriermay be used to refer to a diluent, adjuvant, excipient, or vehicle withwhich the pharmaceutical composition is administered. Water, salinesolutions and aqueous dextrose and glycerol solutions can be employed asliquid carriers, particularly for injectable solutions. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. The formulation should suitthe mode of administration.

The immunogenic composition optionally can comprise one or morephysiologically acceptable buffers selected from, but not limited toTris (trimethamine), phosphate, acetate, borate, citrate, glycine,histidine and succinate. In certain embodiments, the formulation isbuffered to within a pH range of about 5.0 to about 7.0, preferably fromabout 5.5 to about 6.5.

The immunogenic composition optionally can comprise one or morenon-ionic surfactants, including but not limited to polyoxyethylenesorbitan fatty acid esters, Polysorbate-80 (Tween 80), Polysorbate-60(Tween 60), Polysorbate-40 (Tween 40) and Polysorbate-20 (Tween 20),polyoxyethylene alkyl ethers, including but not limited to Brij 58, Brij35, as well as others such as Triton X-100; Triton X-114, NP40, Span 85and the Pluronic series of non-ionic surfactants (e. g., Pluronic 121),with preferred components Polysorbate-80 at a concentration from about0.001% to about 2% (with up to about 0.25% being preferred) orPolysorbate-40 at a concentration from about 0.001% to 1% (with up toabout 0.5% being preferred).

The invention further relates to vaccines comprising the immunogeniccomposition of the invention.

Methods for Inducing an Immune Response and Protecting Against Infection

The present disclosure also includes methods of use for immunogeniccompositions described herein. For example, one embodiment of thedisclosure provides a method of inducing an immune response againstStreptococcus pneumoniae, comprising administering to a subject animmunogenic amount of any of the immunogenic compositions describedherein.

One embodiment of the disclosure provides a method of protecting asubject against an infection with Streptococcus pneumoniae, or a methodof preventing infection with Streptococcus pneumoniae, or a method ofreducing the severity of or delaying the onset of at least one symptomassociated with an infection caused by Streptococcus pneumoniae, themethods comprising administering to a subject an immunogenic amount ofany of the immunogenic compositions described herein.

One embodiment of the disclosure provides a method of protecting asubject against an infection with serotype 10A Streptococcus pneumoniae,or a method of preventing infection with serotype 10A Streptococcuspneumoniae, or a method of reducing the severity of or delaying theonset of at least one symptom associated with an infection caused byserotype 10A Streptococcus pneumoniae, the methods comprisingadministering to a subject an immunogenic amount of any of theimmunogenic compositions described herein.

One embodiment of the disclosure provides a method of treating orpreventing a Streptococcus pneumoniae infection, disease or conditionassociated with serotype 10A Streptococcus pneumoniae in a subject, themethod comprising the step of administering a therapeutically orprophylactically effective amount of an immunogenic compositiondescribed herein to the subject. Another embodiment provides a method oftreating or preventing a Streptococcus pneumoniae infection, disease orcondition associated with a serotype 10A Streptococcus pneumoniae in asubject, the method comprising generating a polyclonal or monoclonalantibody preparation from the immunogenic composition described herein,and using said antibody preparation to confer passive immunity to thesubject.

In one embodiment, the disclosure relates to the use of the immunogenicconjugate or immunogenic composition disclosed herein for themanufacture of a medicament for protecting a subject against aninfection with Streptococcus pneumoniae, and/or preventing infectionwith Streptococcus pneumoniae, and/or reducing the severity of ordelaying the onset of at least one symptom associated with an infectioncaused by Streptococcus pneumoniae, and/or protecting a subject againstan infection with serotype 10A Streptococcus pneumoniae and/orpreventing infection with serotype 10A Streptococcus pneumoniae, and/orreducing the severity of or delaying the onset of at least one symptomassociated with an infection caused by serotype 10A Streptococcuspneumoniae.

One embodiment of the disclosure provides a method of protecting asubject against an infection with serotype 22F Streptococcus pneumoniae,or a method of preventing infection with serotype 22F Streptococcuspneumoniae, or a method of reducing the severity of or delaying theonset of at least one symptom associated with an infection caused byserotype 22F Streptococcus pneumoniae, the methods comprisingadministering to a subject an immunogenic amount of any of theimmunogenic compositions described herein.

One embodiment of the disclosure provides a method of treating orpreventing a Streptococcus pneumoniae infection, disease or conditionassociated with serotype 22F Streptococcus pneumoniae in a subject, themethod comprising the step of administering a therapeutically orprophylactically effective amount of an immunogenic compositiondescribed herein to the subject. Another embodiment provides a method oftreating or preventing a Streptococcus pneumoniae infection, disease orcondition associated with a serotype 22F Streptococcus pneumoniae in asubject, the method comprising generating a polyclonal or monoclonalantibody preparation from the immunogenic composition described herein,and using said antibody preparation to confer passive immunity to thesubject.

In one embodiment, the disclosure relates to the use of the immunogenicconjugate or immunogenic composition disclosed herein for themanufacture of a medicament for protecting a subject against aninfection with Streptococcus pneumoniae, and/or preventing infectionwith Streptococcus pneumoniae, and/or reducing the severity of ordelaying the onset of at least one symptom associated with an infectioncaused by Streptococcus pneumoniae, and/or protecting a subject againstan infection with serotype 22F Streptococcus pneumoniae and/orpreventing infection with serotype 22F Streptococcus pneumoniae, and/orreducing the severity of or delaying the onset of at least one symptomassociated with an infection caused by serotype 22F Streptococcuspneumoniae.

One embodiment of the disclosure provides a method of protecting asubject against an infection with serotype 33F Streptococcus pneumoniae,or a method of preventing infection with serotype 33F Streptococcuspneumoniae, or a method of reducing the severity of or delaying theonset of at least one symptom associated with an infection caused byserotype 33F Streptococcus pneumoniae, the methods comprisingadministering to a subject an immunogenic amount of any of theimmunogenic compositions described herein.

One embodiment of the disclosure provides a method of treating orpreventing a Streptococcus pneumoniae infection, disease or conditionassociated with serotype 33F

Streptococcus pneumoniae in a subject, the method comprising the step ofadministering a therapeutically or prophylactically effective amount ofan immunogenic composition described herein to the subject. Anotherembodiment provides a method of treating or preventing a Streptococcuspneumoniae infection, disease or condition associated with a serotype33F Streptococcus pneumoniae in a subject, the method comprisinggenerating a polyclonal or monoclonal antibody preparation from theimmunogenic composition described herein, and using said antibodypreparation to confer passive immunity to the subject.

In one embodiment, the disclosure relates to the use of the immunogenicconjugate or immunogenic composition disclosed herein for themanufacture of a medicament for protecting a subject against aninfection with Streptococcus pneumoniae, and/or preventing infectionwith Streptococcus pneumoniae, and/or reducing the severity of ordelaying the onset of at least one symptom associated with an infectioncaused by Streptococcus pneumoniae, and/or protecting a subject againstan infection with serotype 33F Streptococcus pneumoniae and/orpreventing infection with serotype 33F Streptococcus pneumoniae, and/orreducing the severity of or delaying the onset of at least one symptomassociated with an infection caused by serotype 33F Streptococcuspneumoniae.

An “immune response” to an immunogenic composition is the development ina subject of a humoral and/or a cell-mediated immune response tomolecules present in the immunogenic composition or vaccine compositionof interest. For purposes of the present disclosure, a “humoral immuneresponse” is an antibody-mediated immune response and involves theinduction and generation of antibodies that recognize and bind with someaffinity for the antigen in the immunogenic composition or vaccine ofthe disclosure, while a “cell-mediated immune response” is one mediatedby T-cells and/or other white blood cells. A “cell-mediated immuneresponse” is elicited by the presentation of antigenic epitopes inassociation with Class I or Class II molecules of the majorhistocompatibility complex (MHC), CD1 or other non-classical MHC-likemolecules. This activates antigen-specific CD4+T helper cells or CD8+cytotoxic T lymphocyte cells (“CTLs”). CTLs have specificity for peptideantigens that are presented in association with proteins encoded byclassical or non-classical MHCs and expressed on the surfaces of cells.CTLs help induce and promote the intracellular destruction ofintracellular microbes, or the lysis of cells infected with suchmicrobes. Another aspect of cellular immunity involves anantigen-specific response by helper T-cells. Helper T-cells act to helpstimulate the function, and focus the activity of, nonspecific effectorcells against cells displaying peptide or other antigens in associationwith classical or non-classical MHC molecules on their surface. A“cell-mediated immune response” also refers to the production ofcytokines, chemokines and other such molecules produced by activatedT-cells and/or other white blood cells, including those derived fromCD4+ and CD8+ T-cells. The ability of a particular antigen orcomposition to stimulate a cell-mediated immunological response may bedetermined by a number of assays, such as by lymphoproliferation(lymphocyte activation) assays, CTL cytotoxic cell assays, by assayingfor T-lymphocytes specific for the antigen in a sensitized subject, orby measurement of cytokine production by T cells in response tore-stimulation with antigen. Such assays are well known in the art. See,e.g., Erickson et al. (1993) J. Immunol. 151:4189-4199; and Doe et al.(1994) Eur. J. Immunol. 24:2369-2376.

As used herein, “treatment” (including variations thereof, e.g., “treat”or “treated”) means any one or more of the following: (i) the preventionof infection or re-infection, as in a traditional vaccine, (ii) thereduction in the severity of, or, in the elimination of symptoms, and(iii) the substantial or complete elimination of the pathogen ordisorder in question. Hence, treatment may be effected prophylactically(prior to infection) or therapeutically (following infection). In thepresent disclosure, prophylactic treatment is the preferred mode.According to a particular embodiment of the present disclosure,compositions and methods are provided that treat, includingprophylactically and/or therapeutically immunize, a host animal againsta serotype 10A Streptococcus pneumoniae infection. According to aparticular embodiment of the present disclosure, compositions andmethods are provided that treat, including prophylactically and/ortherapeutically immunize, a host animal against a serotype 22FStreptococcus pneumoniae infection. According to a particular embodimentof the present disclosure, compositions and methods are provided thattreat, including prophylactically and/or therapeutically immunize, ahost animal against a serotype 33F Streptococcus pneumoniae infection.The methods of the present disclosure are useful for conferringprophylactic and/or therapeutic immunity to a subject. The methods ofthe present disclosure can also be practiced on subjects for biomedicalresearch applications.

An “immunogenic amount,” and “immunologically effective amount,” both ofwhich are used interchangeably herein, refers to the amount of antigenor immunogenic composition sufficient to elicit an immune response,either a cellular (T-cell) or humoral (B-cell or antibody) response, orboth, as measured by standard assays known to one skilled in the art.

In a preferred embodiment, said subject is a human. In a most preferredembodiment, said subject is a newborn (i.e. under three months of age),an infant (from 3 months to one year of age) or a toddler (i.e. from oneyear to four years of age).

In an embodiment, the immunogenic compositions disclosed herein are foruse as a vaccine.

In such embodiment, the subject to be vaccinated may be less than 1 yearof age. For example, the subject to be vaccinated can be about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11 or 12 months of age. In an embodiment, thesubject to be vaccinated is about 2, 4 or 6 months of age. In anotherembodiment, the subject to be vaccinated is less than 2 years of age.For example the subject to be vaccinated can be about 12-15 months ofage. In some cases, as little as one dose of the immunogenic compositionaccording to the invention is needed, but under some circumstances, asecond, third or fourth dose may be given (see regimen section).

In an embodiment of the present invention, the subject to be vaccinatedis a human adult 50 years of age or older, more preferably a human adult55 years of age or older. In an embodiment, the subject to be vaccinatedis a human adult 65 years of age or older, 70 years of age or older, 75years of age or older or 80 years of age or older.

In an embodiment the subject to be vaccinated is an immunocompromisedindividual, in particular a human. An immunocompromised individual isgenerally defined as a person who exhibits an attenuated or reducedability to mount a normal humoral or cellular defense to challenge byinfectious agents.

In an embodiment of the present invention, the immunocompromised subjectto be vaccinated suffers from a disease or condition that impairs theimmune system and results in an antibody response that is insufficientto protect against or treat pneumococcal disease.

In an embodiment, said disease is a primary immunodeficiency disorder.Preferably, said primary immunodeficiency disorder is selected from thegroup consisting of: combined T- and B-cell immunodeficiencies, antibodydeficiencies, well-defined syndromes, immune dysregulation diseases,phagocyte disorders, innate immunity deficiencies, autoinflammatorydisorders, and complement deficiencies. In an embodiment, said primaryimmunodeficiency disorder is selected from the one disclosed on page 24line 11 to page 25 line 19 of the PCT application WO2010/125480.

In a particular embodiment of the present invention, theimmunocompromised subject to be vaccinated suffers from a diseaseselected from the groups consisting of: HIV-infection, acquiredimmunodeficiency syndrome (AIDS), cancer, chronic heart or lungdisorders, congestive heart failure, diabetes mellitus, chronic liverdisease, alcoholism, cirrhosis, spinal fluid leaks, cardiomyopathy,chronic bronchitis, emphysema, Chronic obstructive pulmonary disease(COPD), spleen dysfunction (such as sickle cell disease), lack of spleenfunction (asplenia), blood malignancy, leukemia, multiple myeloma,Hodgkin's disease, lymphoma, kidney failure, nephrotic syndrome andasthma.

In an embodiment of the present invention, the immunocompromised subjectto be vaccinated suffers from malnutrition.

In a particular embodiment of the present invention, theimmunocompromised subject to be vaccinated is taking a drug or treatmentthat lowers the body's resistance to infection. In an embodiment, saiddrug is selected from the one disclosed on page 26 line 33 to page 26line 40 of the PCT application WO2010/125480.

In a particular embodiment of the present invention, theimmunocompromised subject to be vaccinated is a smoker.

In a particular embodiment of the present invention, theimmunocompromised subject to be vaccinated has a white blood cell count(leukocyte count) below 5×10⁹ cells per liter, or below 4×10⁹ cells perliter, or below 3×10⁹ cells per liter, or below 2×10⁹ cells per liter,or below 1×10⁹ cells per liter, or below 0.5×10⁹ cells per liter, orbelow 0.3×10⁹ cells per liter, or below 0.1×10⁹ cells per liter.

White blood cell count (leukocyte count): The number of white bloodcells (WBCs) in the blood. The WBC is usually measured as part of theCBC (complete blood count). White blood cells are the infection-fightingcells in the blood and are distinct from the red (oxygen-carrying) bloodcells known as erythrocytes. There are different types of white bloodcells, including neutrophils (polymorphonuclear leukocytes; PMNs), bandcells (slightly immature neutrophils), T-type lymphocytes (T cells),B-type lymphocytes (B cells), monocytes, eosinophils, and basophils. Allthe types of white blood cells are reflected in the white blood cellcount. The normal range for the white blood cell count is usuallybetween 4,300 and 10,800 cells per cubic millimeter of blood. This canalso be referred to as the leukocyte count and can be expressed ininternational units as 4.3-10.8×10⁹ cells per liter.

In a particular embodiment of the present invention, theimmunocompromised subject to be vaccinated suffers from neutropenia. Ina particular embodiment of the present invention, the immunocompromisedsubject to be vaccinated has a neutrophil count below 2×10⁹ cells perliter, or below 1×10⁹ cells per liter, or below 0.5×10⁹ cells per liter,or below 0.1×10⁹ cells per liter, or below 0.05×10⁹ cells per liter.

A low white blood cell count or “neutropenia” is a conditioncharacterized by abnormally low levels of neutrophils in the circulatingblood. Neutrophils are a specific kind of white blood cell that helpprevent and fight infections. The most common reason that cancerpatients experience neutropenia is as a side effect of chemotherapy.Chemotherapy-induced neutropenia increases a patient's risk of infectionand disrupts cancer treatment.

In a particular embodiment of the present invention, theimmunocompromised subject to be vaccinated has a CD4+ cell count below500/mm³, or CD4+ cell count below 300/mm³, or CD4+ cell count below200/mm³, CD4+ cell count below 100/mm³, CD4+ cell count below 75/mm³, orCD4+ cell count below 50/mm³.

CD4 cell tests are normally reported as the number of cells in mm³.Normal CD4 counts are between 500 and 1600, and CD8 counts are between375 and 1100. CD4 counts drop dramatically in people with HIV.

In an embodiment of the invention, any of the immunocompromised subjectdisclosed herein is a human male or a human female.

The amount of a conjugate in a composition is generally calculated basedon total polysaccharide, conjugated and non-conjugated for thatconjugate. For example, a conjugate with 20% free polysaccharide willhave about 80 mcg of conjugated polysaccharide and about 20 mcg ofnon-conjugated polysaccharide in a 100 mcg polysaccharide dose. Theprotein contribution to the conjugate is usually not considered whencalculating the dose of a conjugate. Generally, each dose will comprise0.1 to 100 mcg of polysaccharide, particularly 0.1 to 10 mcg, and moreparticularly 1 to 10 mcg and more particularly 1 to 5 μg. Preferablyeach dose will comprise about 1.1, 2, 2.2, 3, 3.3, 4, 4.4 μg ofpolysaccharide.

Optimal amounts of components for a particular immunogenic compositionor vaccine can be ascertained by standard studies involving observationof appropriate immune responses in subjects. Following an initialvaccination, subjects can receive one or several booster immunizationsadequately spaced.

The effectiveness of an antigen as an immunogen, can be measured eitherby proliferation assays, by cytolytic assays, such as chromium releaseassays to measure the ability of a T-cell to lyse its specific targetcell, or by measuring the levels of B-cell activity by measuring thelevels of circulating antibodies specific for the antigen in serum. Animmune response may also be detected by measuring the serum levels ofantigen specific antibody induced following administration of theantigen, and more specifically, by measuring the ability of theantibodies so induced to enhance the opsonophagocytic ability ofparticular white blood cells, as described herein. The level ofprotection of the immune response may be measured by challenging theimmunized host with the antigen that has been administered. For example,if the antigen to which an immune response is desired is a bacterium,the level of protection induced by the immunogenic amount of the antigenis measured by detecting the percent survival or the percent mortalityafter challenge of the animals with the bacterial cells. In oneembodiment, the amount of protection may be measured by measuring atleast one symptom associated with the bacterial infection, e.g., a feverassociated with the infection. The amount of each of the antigens in themulti-antigen or multi-component vaccine or immunogenic compositionswill vary with respect to each of the other components and can bedetermined by methods known to the skilled artisan. Such methods wouldinclude procedures for measuring immunogenicity and/or in vivo efficacy.

The disclosure further provides antibodies and antibody compositionswhich bind specifically and selectively to the capsular polysaccharidesor glycoconjugates of the present disclosure. In some embodiments,antibodies are generated upon administration to a subject of thecapsular polysaccharides or glycoconjugates of the present disclosure.In some embodiments, the disclosure provides purified or isolatedantibodies directed against one or more of the capsular polysaccharidesor glycoconjugates of the present disclosure. In some embodiments, theantibodies of the present disclosure are functional as measured bykilling bacteria in either an animal efficacy model or via anopsonophagocytic killing assay. In some embodiments, the antibodies ofthe disclosure confer passive immunity to a subject. The presentdisclosure further provides polynucleotide molecules encoding anantibody or antibody fragment of the disclosure, and a cell, cell line(such as hybridoma cells or other engineered cell lines for recombinantproduction of antibodies) or a transgenic animal that produces anantibody or antibody composition of the disclosure, using techniqueswell-known to those of skill in the art.

EXAMPLES Example 1: Preparation of Serotype 22F Polysaccharide—CRM₁₉₇Conjugate

1.1. Preparation of Isolated Streptococcus pneumoniae Serotype 22FPolysaccharide

Serotype 22F capsular polysaccharides can be obtained directly frombacteria using isolation procedures known to one of ordinary skill inthe art. (see for example methods disclosed U.S. Patent App. Pub. Nos.20060228380, 20060228381, 20070184071, 20070184072, 20070231340, and20080102498 or WO2008118752). The serotype 22F Streptococcus pneumoniawere grown in a seed bottle and then transferred to a seed fermentor.Once the targeted optical density was reached, the cells weretransferred to a production fermentor. The fermentation was broth wasinactivated by the addition of N-lauroyl sarcosine and purified byultrafiltration and diafiltration.

The purified Streptococcus pneumoniae serotype 22F polysaccharide wassized by high pressure homogenization using a PANDA 2K homogenizer (GEANiro Soavi) to produce the isolated Streptococcus pneumoniae serotype22F polysaccharide.

1.2. Oxidation of Isolated Streptococcus pneumoniae Serotype 22FCapsular Polysaccharide

Oxidation of Polysaccharide was carried out in 100 mM potassiumphosphate buffer (pH 5.8±0.2) obtained by sequential addition ofcalculated amount of 500 mM potassium phosphate buffer (pH 5.8) and WFIto give final polysaccharide concentration of 2.0 g/L. If required, thereaction pH was adjusted to 5.8, approximately. After pH adjustment, thereaction temperature was lowered to 5±3° C. Oxidation was initiated bythe addition of 0.10±0.02 molar equivalents (MEq) of sodium periodate.The target oxidation reaction time is 16±1 hrs at 5±3° C.

The oxidation reaction was quenched with 2 MEq of 2,3-butanediol undercontinuous stirring at 5±3° C. for 1-2 hrs.

Concentration and diafiltration of the activated polysaccharide wascarried out using 100K MWCO ultrafiltration cassettes. Diafiltration wasperformed against 35-fold diavolume of WFI. The purified activatedpolysaccharide was stored at 5±3° C. The purified activated saccharideis characterized inter alia by (i) Molecular Weight by SEC-MALLS (ii)presence of O-acetyl and (iii) Degree of Oxidation.

SEC-MALLS is used for the determination of the molecular weight ofpolysaccharides and polysaccharide-protein conjugates. SEC is used toseparate the polysaccharides by hydrodynamic volume. Refractive index(RI) and multi-angle laser light scattering (MALLS) detectors are usedfor the determination of the molecular weight. When light interacts withmatter, it scatters and the amount of scattered light is related to theconcentration, the square of the do/dc (the specific refractive indexincrements), and the molar mass of the matter. The molecular weightmeasurement is calculated based on the readings from the scattered lightsignal from the MALLS detector and the concentration signal from the RIdetector.

The degree of oxidation (DO=moles of sugar repeat unit/moles ofaldehyde) of the activated polysaccharide was determined as follows:

The moles of sugar repeat unit is determined by various colorimetricmethods, such as for example by using Anthrone method. Thepolysaccharide is first broken down to monosaccharides by the action ofsulfuric acid and heat. The Anthrone reagent reacts with the hexoses toform a yellow-green colored complex whose absorbance is readspectrophotometrically at 625 nm. Within the range of the assay, theabsorbance is directly proportional to the amount of hexose present.

The moles of aldehyde also is determined simultaneously, using MBTHcolorimetric method. The MBTH assay involves the formation of an azinecompound by reacting aldehyde groups (from a given sample) with a3-methyl-2-benzothiazolone hydrazone (MBTH assay reagent). The excess3-methyl-2-benzothiazolone hydrazone oxidizes to form a reactive cation.The reactive cation and the azine react to form a blue chromophore. Theformed chromophore is then read spectroscopically at 650 nm.

1.3. Conjugation an Activated Streptococcus pneumoniae Serotype 22FPolysaccharide with CRM₁₉₇

The conjugation process consists of the following steps:

a. Compounding with sucrose excipient, and lyophilization.b. Reconstitution of the lyophilized polysaccharide and CRM₁₉₇.c. Conjugation of activated polysaccharide to CRM₁₉₇ and cappingd. Purification of the conjugatea. Compounding with Sucrose and Lyophilization

The activated polysaccharide was compounded with sucrose (50% w/v inWFI) to a ratio of 25 grams of sucrose per gram of activatedpolysaccharide. The bottle of compounded mixture was then lyophilized.Following lyophilization, bottles containing lyophilized activatedpolysaccharide were stored at −20±5° C. Calculated amount of CRM₁₉₇protein (target S/P input ratio=1) was shellfrozen and lyophilizedseparately. Lyophilized CRM₁₉₇ was stored at −20±5° C.

b. Reconstitution of Lyophilized Activated Polysaccharide and CRM₁₉₇Protein

Lyophilized activated polysaccharide was reconstituted in anhydrousdimethyl sulfoxide (DMSO). Upon complete dissolution of polysaccharide,an equal amount of anhydrous

DMSO was added to lyophilized CRM₁₉₇ for reconstitution.

c. Conjugation of Activated Polysaccharide to CRM₁₉₇ and Capping

Reconstituted CRM₁₉₇ (in DMSO) was combined in the conjugation reactionvessel with the reconstituted activated polysaccharide. The finalpolysaccharide concentration in reaction solution is 1 g/L. Conjugationwas initiated by adding 1.5±0.1 MEq of sodium cyanoborohydride to thereaction mixture and the reaction was incubated at 23±2° C. for 20±2hrs. Termination of conjugation reaction is done by adding 2 MEq ofsodium borohydride. The capping reaction was incubated at 23±2° C. for3±1 hrs.

d. Purification of Conjugate

The conjugate solution was diluted 1:5 with chilled 5 mM succinate-0.9%saline (pH 6.0) in preparation for purification by tangential flowfiltration using 100K MWCO membranes and a 20× diafiltration wasperformed using 5 mM succinate-0.9% saline (pH6.0) as the medium. Afterthe diafiltration was completed, the conjugate retentate was furtherdiluted, filtered through a 0.22 μm filter and stored at 2-8° C.

Table 1 comprises characterizing data for serotype 22Fpolysaccharide-CRM₁₉₇ conjugates obtained by the method of theinvention. In particular, conjugates 5 and 6 of the table were obtainedas disclosed in example 1.

Serotype 22F polysaccharide-CRM₁₉₇ conjugates generated by the RAC-DMSOprocess provided better yields and exhibited better batch to batchconsistency in the MW along with significantly lower % Free saccharidelevels and higher carrier protein (Lysine) modification compared to theconjugates generated by the RAC-aqueous process, as shown in Table 1.

TABLE 1 Serotype 22F Conjugates Conjugate No 1 2 3 4 5 6 Aqueous AqueousAqueous DMSO DMSO DMSO Act Poly 488 803 410 639 709 416 MW, kDa DO 17.315 10.3 14.6 14.4 13.7 Input Ratio 0.8 0.8 1 0.8 1 1 % Conjugate Yield66 63 46 70 62 75 % Free Saccharide 18 43 45 <5 <5 8 Conjugate MW 10243544 462 3911 3734 4453 Saccharide-Protein 1.6 1.1 1.45 0.8 0.65 1.0Ratio Modified Lys (AAA) 2.3 N/A N/A 6.1 N/A 4.7 % Conjugate yield iscalculated as follows: (amount of polysaccharide in the conjugate×100)/amount of activated polysaccharide.

Example 2: Preparation of Serotype 10A Polysaccharide—CRM₁₉₇ Conjugate

2.1. Preparation of Isolated Streptococcus pneumoniae Serotype 10APolysaccharide

Isolated Serotype 10A polysaccharides was obtained as disclosed inexample 1.1 except that the purified polysaccharide was not sized.

2.2. Oxidation of Isolated Streptococcus pneumoniae Serotype 10ACapsular Polysaccharide

A calculated volume of 0.1M potassium phosphate buffer (pH 6.0) andwater-for-injection (WFI) was added to the polysaccharide solution toachieve a final polysaccharide concentration of 2.5 g/L and a finalconcentration of 25 mM potassium phosphate buffer, If required pH wasadjusted to 6.0, approximately. The diluted polysaccharide was thencooled to 5±3° C. Oxidation was initiated by the addition of 0.25±0.02molar equivalents (MEq) of sodium periodate solution. The oxidationreaction time was approximately 4 hrs at 5±3° C. The oxidation reactionwas quenched with 1 MEq of 2,3-butanediol under continuous stirring at5±3° C. for 1-2 hrs.

After reaching the target reaction time, the activated polysaccharidewas concentrated using 30K MWCO Millipore ultrafiltration cassettes. Thediafiltration was then performed against 20-fold diavolume of WFI. Thepurified activated polysaccharide was stored at 5±3° C. The purifiedactivated saccharide is characterized inter alia by (i) Molecular Weightby SEC-MALLS and (ii) Degree of Oxidation.

2.3 Conjugation an Activated Streptococcus pneumoniae Serotype 10APolysaccharide with CRM₁₉₇

The conjugation process consists of the following steps:

a. Compounding with sucrose excipient, and lyophilization.b. Reconstitution of the lyophilized polysaccharide and CRM197.c. Conjugation of activated polysaccharide to CRM197 and cappingd. Purification of the conjugatea. Compounding with Sucrose

The activated polysaccharide is compounded with sucrose to a ratio of25±2.5 grams of sucrose per gram of activated polysaccharide. The bottleof compounded mixture was then lyophilized. Following lyophilization,bottles containing lyophilized activated polysaccharide were stored at−20±5° C.

b. Reconstitution of Lyophilized Activated Polysaccharide and CRM₁₉₇Protein

Lyophilized activated polysaccharide was reconstituted in anhydrousdimethyl sulfoxide (DMSO). Upon complete dissolution of polysaccharide,the same amount of anhydrous DMSO was added to the calculated CRM₁₉₇ forreconstitution.

c. Conjugation of Activated Polysaccharide to CRM₁₉₇ and Capping

Reconstituted CRM₁₉₇ (in DMSO) was added to the reconstituted activatedpolysaccharide in the conjugation reactor. The final polysaccharideconcentration is 1 g/L. Conjugation was performed by adding 1.2±0.1 MEqof sodium cyanoborohydride to the reaction mixture. The reaction wasincubated and at 23±2° C. for 24±2 hrs. Termination of conjugationreaction is done by adding 2 MEq of sodium borohydride. The cappingreaction was incubated at 23±2° C. for 3±1 hrs.

Termination of conjugation reaction is done by adding 2 MEq of sodiumborohydride. This capping reaction proceeded for 3±1 hrs at 23±2° C.

d. Purification of Conjugate

The conjugate solution was then diluted into 5× (by volume) chilled 5 mMsuccinate-0.9% saline (pH 6.0 and a 20× diafiltration was performedusing 5 mM succinate-0.9% saline (pH6.0). After the initialdiafiltration was completed, the conjugate retentate was transferredthrough a 0.22 μm filter. The conjugate was diluted further with 5 mMsuccinate/0.9% saline (pH 6), and after the final 0.22 μm filtrationstep it was stored at 2-8° C.

Table 2 comprises characterizing data for serotype 10Apolysaccharide-CRM₁₉₇ conjugates obtained by the method of theinvention. In particular, conjugates 4 to 6 of table 2 were obtained asdisclosed in example 2.

Serotype 10A polysaccharide-CRM₁₉₇ conjugates generated by the RAC-DMSOprocess provided better yields and exihibited better batch to batchconsistency in the MW along with significantly lower % Free saccharidelevels and higher carrier protein (Lysine) modification compared to theconjugates generated by the RAC-aqueous process, as shown in Table 2.

TABLE 2 Serotype 10A Conjugates Conjugate No 1 2 3 4 5 6 Aqueous AqueousAqueous DMSO DMSO DMSO DO 12.2 12.2 12.8 10.3 10.8 10.5 Activated 157157 163 170 170 170 Saccharide MW, kda Input Ratio 1.0 1.0 1.0 1.1 1.11.1 % Yield 71 34 50 82 73 66 % Free Saccharide 20 31 26 6.8 6.4 9.7Conjugate MW, kDa 1550 968 3050 4034 3463 5540 Saccharide-protein 1.62.6 1.6 1.1 1.2 1.0 ratio Lys modification 2.6 N/A N/A 6.9 6.7 6.1 AAA

Example 3: Preparation of Serotype 33F Polysaccharide—CRM₁₉₇ Conjugate

3.1. Preparation of Isolated Streptococcus pneumoniae Serotype 33FPolysaccharide

Isolated Serotype 33F polysaccharide was obtained as disclosed inexample 1.1.

3.2. Oxidation of Isolated Streptococcus pneumoniae Serotype 33FCapsular Polysaccharide

A calculated volume of 0.1M sodium phosphate buffer (pH 6.0) andwater-for-injection (WFI) was added to the polysaccharide solution toachieve a final polysaccharide concentration of 2 g/L. If required pHwas adjusted to 6.0, approximately. The diluted polysaccharide was thencooled to 5±3° C. Oxidation was initiated by the addition of 0.1 molarequivalent (MEq) of sodium periodate solution. The oxidation reactiontime was approximately 20 hrs at 5±3° C. The oxidation reaction wasquenched with 1 MEq of 2,3-butanediol under continuous stirring at 5±3°C. for about 1 hour. After reaching the target reaction time, theactivated polysaccharide was concentrated using 100K MWCO Milliporeultrafiltration cassettes. The diafiltration was then performed against40-fold diavolume of WFI. The purified activated polysaccharide wasstored at 5±3° C. The purified activated saccharide is characterizedinter alia by (i) Molecular Weight by SEC-MALLS and (ii) Degree ofOxidation.

3.3 Conjugation an Activated Streptococcus pneumoniae Serotype 33FPolysaccharide with CRM₁₉₇

The serotype 33F polysaccharide-CRM197 conjugate was obtained by aprocess similar to the process of example 1.3, using the activated 33Fpolysaccharide obtained in example 3.2.

Serotype 33F polysaccharide-CRM₁₉₇ conjugates generated by the RAC-DMSOprocess provided better yields and exhibited better batch to batchconsistency in terms of preservation of O-Acetyl levels along withoverall lower % Free saccharide levels and higher carrier protein(Lysine) modification compared to the conjugates generated by theRAC-aqueous process, as shown in Table 3.

TABLE 3 Serotype 33F Conjugates Conjugate No 1 2 3 4 5 6 Aq Aq Aq DMSODMSO DMSO DO 15.2 15.5 15.7 17 9 14 Activated Poly MW 1126 573 574 120328 128 % Yield 65 51 48 78 86 66 % Free Saccharide 8.5 19 13 18 5 <5Conjugate MW, kDa 1341 2137 3674 3158 2165 2001 Saccharide-protein 1.71.4 1.3 1.1 1.1 0.79 ratio % O—Ac preserved <LOQ <LOQ <LOQ 82 100% 100%Conj Lys (AAA) N/A 2.3 2.3 4.5 3.4 5.2 LOQ = Limit of Quantitation

Example 4: Effect of Quenching on Molecular Weight of Activated Serotype22F and 33F Polysaccharides

Activation of 22F and 33F polysaccharides was carried out at either 4°C. or 22° C. Both temperatures gave similar DO values. However, due tothe fact that elevated temperature resulted in faster breakdown ofactivated polysaccharide thereby lowering activated poly MW (as shownwhen comparing for example FIG. 4 (22° C.) and FIG. 5 (4° C.)), 4° C.was generally preferred for activation reaction. In addition, activationdata showed that quenching of un-reacted NalO₄ after oxidation is anessential step for activation, especially for activation using higheramount of NalO₄. The graph shown in FIG. 6 indicate that the MW of theactivated 33F polysaccharide is relatively stable when increasingconcentrations of periodate are used for activation (with quenching)while such MW is highly variable when no quenching step is used. Atspecific conditions set up to obtain a targeted degree of oxidation, themolecular weight of the activated polysaccharide is less variable whenthe quenching step is used. Addition of quenching reagent afteroxidation helps to keep the structural integrity of activatedpolysaccharide until the completion of purification for example byultrafiltration and diafiltration.

Example 5: Opsonophagocytic Activity Assay (OPA)

The immunogenicity of the conjugates obtained by the processes disclosedherein can be assessed using the opsonophagocytic assay (OPA) describedbelow.

Groups of thirty 6-7 week old female Swiss Webster mice were immunizedwith 0.001 pg, 0.01 pg, or 0.1 pg of test conjugates via thesubcutaneous route on week 0. The mice were boosted with the same doseof conjugate on week 3 and then bled at week 4. Serotype-specific OPAswere performed on week 4 sera samples.

Validated opsonophagocytic activity (OPA) assays are used to measurefunctional antibodies in murine sera specific for S. pneumonia serotype10A, 22F or 33F. Test serum is set up in assay reactions that measurethe ability of capsular polysaccharide specific immunoglobulin toopsonize bacteria, trigger complement deposition, thereby facilitatingphagocytosis and killing of bacteria by phagocytes. The OPA titer isdefined as the reciprocal dilution that results in a 50% reduction inbacterial count over control wells without test serum. The OPA titer isinterpolated from the two dilutions that encompass this 50% killingcut-off.

OPA procedures were based on methods described in Hu et al., Clin DiagnLab Immunol 2005; 12(February (2)):287-95 with the followingmodifications. Test serum was serially diluted 2.5-fold and added tomicrotiter assay plates. Live serotype 10A, 22F and 33F target bacterialstrains were added to the wells and the plates were shaken at either 25°C. (serotype 22F) or 37° C. (serotype 10A and 33F) for 30 minutes.Differentiated HL-60 cells (phagocytes) and baby rabbit serum (3- to4-week old, Pel-Freez®, 12.5% final concentration) were added to thewells, and the plates were shaken at 37° C. for 45 minutes (serotype 22Fand 33F) or 60 minutes (serotype 10A). To terminate the reaction, 80 μLof 0.9% NaCl was added to all wells, mixed, and a 10 μL aliquot weretransferred to the wells of MultiScreen HTS HV filter plates(Millipore®) containing 200 μL of water. Liquid was filtered through theplates under vacuum, and 150 μL of HySoy medium was added to each welland filtered through. The filter plates were then incubated at 37° C.,5% CO₂ overnight and were then fixed with Destain Solution (Bio-Rad).The plates were then stained with Coomassie Blue and destained once.Colonies were imaged and enumerated on a Cellular Technology Limited(CTL) ImmunoSpot Analyzer®. Raw colony counts were used to plot killcurves and calculate OPA titers.

Serotype 10A polysaccharide-CRM₁₉₇ conjugates, Serotype 22Fpolysaccharide-CRM₁₉₇ conjugates and Serotype 33F polysaccharide-CRM₁₉₇conjugates obtained as disclosed in examples 1 to 3 were tested in theOPA assay disclosed above and were found to be immunogenic.

1. A process for preparing an activated Streptococcus pneumoniae serotype 10A, 22F or 33F capsular polysaccharide, the process comprising the steps of: (a) reacting an isolated serotype 10A, 22F or 33F capsular polysaccharide with an oxidizing agent; and (b) quenching the oxidation reaction by addition of a quenching agent resulting in an activated Streptococcus pneumoniae serotype 10A, 22F or 33F polysaccharide.
 2. A process according to claim 1 wherein the concentration of serotype 10A, 22F or 33F polysaccharide in step (a) is between 0.5 and 5 mg/mL.
 3. A process according to claim 1 or 2 wherein said polysaccharide is reacted with 0.01 to 10, 0.05 to 5, 0.1 to 1, 0.5 to 1, 0.7 to 0.8, 0.01 to 0.2, 0.05 to 0.5, 0.05 to 0.2, 0.1 to 0.3 molar equivalents of oxidizing agent in step (a).
 4. A process according to any one of claims 1 to 3 wherein said polysaccharide is reacted with 0.05 to 0.3 molar equivalents of oxidizing agent in step (a).
 5. A process according to any one of claims 1 to 4 where the oxidizing agent is periodate.
 6. A process according to any one of claims 1 to 4 where the oxidizing agent is sodium metaperiodate.
 7. A process according to any one of claims 1 to 6 wherein the temperature of the reaction in step (a) is maintained between 1 and 10° C.
 8. A process according to any one of claims 1 to 7 wherein the temperature of the reaction in step (a) is about ° C. or about 5° C.
 9. A process according to any one of claims 1 to 8 wherein the duration of step (a) is between 1 and 25 hours.
 10. A process according to any one of claims 1 to 9 where the quenching agent is selected from vicinal diols, 1,2-aminoalcohols, amino acids, glutathione, sulfite, bisulfate, dithionite, metabisulfite, thiosulfate, phosphites, hypophosphites or phosphorous acid.
 11. A process according to any one of claims 1 to 9 where the quenching agent is a compound of formula (II)

wherein R¹ and R² are each independently selected from H, methyl, ethyl, propyl or isopropyl.
 12. A process according to any one of claims 1 to 9 where the quenching agent is selected from glycerol, ethylene glycol, propan-1,2-diol, butan-1,2-diol or butan-2,3-diol.
 13. A process according to any one of claims 1 to 9 where the quenching agent is butan-2,3-diol.
 14. A process according to any one of claims 1 to 13 wherein the oxidation reaction is quenched by the addition of 0.1 to 10, 0.5 to 5, 0.5 to 3 or 0.5 to 2 molar equivalents of quenching agent.
 15. A process according to any one of claims 1 to 13 wherein the oxidation reaction is quenched by the addition of 0.5 to 2 molar equivalents of quenching agent.
 16. A process according to any one of claims 1 to 15 wherein the temperature of the reaction in step (b) is maintained between 1 and 10′C.
 17. A process according to any one of claims 1 to 16 wherein the temperature of the reaction in step (b) is about 4° C.
 18. A process according to any one of claims 1 to 17 wherein the duration of step (b) is between 0.5 and 2 hours.
 19. A process according to any one of claims 1 to 18 wherein the activated polysaccharide is purified after step (b).
 20. A process according to any one of claims 1 to 19 wherein said isolated polysaccharide is an isolated Streptococcus pneumoniae serotype 10A polysaccharide.
 21. A process according to claim 20 wherein said isolated Streptococcus pneumoniae serotype 10A polysaccharide is obtained by a process comprising the steps of: (a) preparing a fermentation culture of serotype 10A Streptococcus pneumonia bacterial cells; (b) lysing the bacterial cells in said fermentation culture; and (c) purifying serotype 10A polysaccharide from the fermentation culture.
 22. An activated Streptococcus pneumoniae serotype 10A capsular polysaccharide obtained by a process of any one of claims 1 to
 21. 23. An activated Streptococcus pneumoniae serotype 10A capsular polysaccharide obtainable by a process of any one of claims 1 to
 21. 24. An activated Streptococcus pneumoniae serotype 10A capsular polysaccharide according to any one of claim 22 or 23, where said polysaccharide has a molecular weight between 50 and 400, 50 and 350, 50 and 300, 50 and 250, 50 and 200, 100 and 300, 100 and 250, or 100 and 200 kDa.
 25. An activated Streptococcus pneumoniae serotype 10A capsular polysaccharide according to any one of claim 22 or 23, where said polysaccharide has a molecular weight between 50 and 300 kDa.
 26. An activated Streptococcus pneumoniae serotype 10A capsular polysaccharide according to any one of claim 22 or 23, where said polysaccharide has a molecular weight between 100 and 200 kDa.
 27. An activated Streptococcus pneumoniae serotype 10A capsular polysaccharide according to any one of claims 22 to 26, where said polysaccharide has a degree of oxidation between 5 and 20, 5 and 15, 8 and 14, 8 and 12, or 9 and
 11. 28. An activated Streptococcus pneumoniae serotype 10A capsular polysaccharide according to any one of claims 22 to 26, where said polysaccharide has a degree of oxidation between 9 and
 11. 29. A process according to any one of claims 1 to 19 wherein said isolated polysaccharide is an isolated Streptococcus pneumoniae serotype 22F polysaccharide.
 30. A process according to claim 29 wherein said isolated Streptococcus pneumoniae serotype 22F polysaccharide is obtained by a process comprising the steps of: (a) preparing a fermentation culture of serotype 22F Streptococcus pneumonia bacterial cells; (b) lysing the bacterial cells in said fermentation culture; (c) purifying serotype 22F polysaccharide from the fermentation culture; and (d) sizing the serotype 22F polysaccharide by mechanical sizing, preferably by high pressure homogenization.
 31. An activated Streptococcus pneumoniae serotype 22F capsular polysaccharide obtained by a process of any one of claim 1 to 19 or 29 or
 30. 32. An activated Streptococcus pneumoniae serotype 22F capsular polysaccharide obtainable by a process of any one of claim 1 to 19 or 29 or
 30. 33. An activated Streptococcus pneumoniae serotype 22F capsular polysaccharide according to any one of claim 31 or 32, where said polysaccharide has a molecular weight between 25 and 1000, 100 and 1000, 300 and 800, 300 and 700, 300 and 600, 400 and 1000, 400 and 800, 400 and 700, or 400 and 600 kDa.
 34. An activated Streptococcus pneumoniae serotype 22F capsular polysaccharide according to any one of claim 31 or 32, where said polysaccharide has a molecular weight between 300 and 800 kDa.
 35. An activated Streptococcus pneumoniae serotype 22F capsular polysaccharide according to any one of claim 31 or 32, where said polysaccharide has a molecular weight between 400 and 600 kDa.
 36. An activated Streptococcus pneumoniae serotype 22F capsular polysaccharide according to any one of claims 31 to 35, where said polysaccharide has a degree of oxidation between 10 and 25, 10 and 20, 12 and 20, or 14 and
 18. 37. An activated Streptococcus pneumoniae serotype 22F capsular polysaccharide according to any one of claims 31 to 35, where said polysaccharide has a degree of oxidation between 10 and
 20. 38. An activated Streptococcus pneumoniae serotype 22F capsular polysaccharide according to any one of claims 31 to 37, where said polysaccharide comprises at least 0.6 mM acetate per mM serotype 22F polysaccharide.
 39. A process according to any one of claims 1 to 19 wherein said isolated polysaccharide is an isolated Streptococcus pneumoniae serotype 33F polysaccharide.
 40. A process according to claim 39 wherein said isolated Streptococcus pneumoniae serotype 33F polysaccharide is obtained by a process comprising the steps of: (a) preparing a fermentation culture of serotype 33F Streptococcus pneumonia bacterial cells; (b) lysing the bacterial cells in said fermentation culture; (c) purifying serotype 33F polysaccharide from the fermentation culture; and (d) sizing the serotype 33F polysaccharide by mechanical sizing, preferably by high pressure homogenization.
 41. An activated Streptococcus pneumoniae serotype 33F capsular polysaccharide obtained by a process of any one of claim 1 to 19 or 39 or
 40. 42. An activated Streptococcus pneumoniae serotype 33F capsular polysaccharide obtainable by a process of any one of claim 1 to 19 or 39 or
 40. 43. An activated Streptococcus pneumoniae serotype 33F capsular polysaccharide according to claim 39 or 40, where said polysaccharide has a molecular weight between 50 and 1250, 200 and 1200, 500 and 1200, 500 and 1000, 700 and 1200, 800 and 1200, 800 and 1100, 900 and 1200, or 800 and 1000 Da.
 44. An activated Streptococcus pneumoniae serotype 33F capsular polysaccharide according to claim 39 or 40, where said polysaccharide has a molecular weight between 500 and 1000 kDa.
 45. An activated Streptococcus pneumoniae serotype 33F capsular polysaccharide according to claim 39 or 40, where said polysaccharide has a molecular weight between 50 and 400 kDa.
 46. An activated Streptococcus pneumoniae serotype 33F capsular polysaccharide according to any one of claims 41 to 45, where said polysaccharide has a degree of oxidation between 10 and
 20. 47. An activated Streptococcus pneumoniae serotype 33F capsular polysaccharide according to any one of claims 41 to 34, where said polysaccharide comprises at least 0.7 mM acetate per mM serotype 33F polysaccharide.
 48. A process according to any one of claims 1 to 47 wherein the activated polysaccharide is lyophilized after step (b).
 49. A process for the preparation of an immunogenic conjugate comprising Streptococcus pneumoniae serotype 10A, 22F or 33F polysaccharide covalently linked to a carrier protein, the process comprising the steps of: (a) compounding an activated serotype 10A, 22F or 33F polysaccharide obtained or obtainable by a process of any one of claims 1 to 19 with a carrier protein; and, (b) reacting the compounded, activated serotype 10A, 22F or 33F polysaccharide and carrier protein with a reducing agent to form a serotype 10A, 22F or 33F polysaccharide:carrier protein conjugate.
 50. A process according to claim 49, said process further comprising the step of: (c) capping unreacted aldehydes in the serotype 10A, 22F or 33F polysaccharide:carrier protein conjugate.
 51. A process according to claim 49 or 50 where step (a) and (b) are carried out in DMSO.
 52. A process according to claim 49 or 50 where step (a) and (b) are carried out in an aqueous solution.
 53. A process according to anyone of claims 49 to 52 where the activated polysaccharide and the protein carrier are co-lyophilized after step (a).
 54. A process according to claim 53 where step (b) is carried out in DMSO.
 55. A process according to claim 53 where step (b) is carried out in an aqueous solution.
 56. A process according to anyone of claims 49 to 55 where the concentration of activated polysaccharide in step (b) is between 0.1 and 10 mg/mL, 0.5 and 5 mg/mL, or 0.5 and 2 mg/mL.
 57. A process according to anyone of claims 49 to 55 where the concentration of activated polysaccharide in step (b) is between 0.5 and 2 mg/mL.
 58. A process according to anyone of claims 49 to 57 where the initial ratio of activated serotype 10A, 22F or 33F polysaccharide to carrier protein is between 5:1 and 0.1:1, 2:1 and 0.1:1, 2:1 and 1:1, 1.5:1 and 1:1, 0.1:1 and 1:1, 0.3:1 and 1:1, or 0.6:1 and 1.2:1.
 59. A process according to anyone of claims 49 to 57 where the initial ratio of activated serotype 10A, 22F or 33F polysaccharide to carrier protein is between 0.6:1 and 1.2:1.
 60. A process according to anyone of claims 49 to 59 where, in step (b), the activated polysaccharide is reacted with between 0.5 to 2.5 molar equivalents of reducing agent.
 61. A process according to anyone of claims 49 to 60 where the reducing agent is sodium cyanoborohydride.
 62. A process according to anyone of claims 49 to 61 wherein the duration of step (d) is between 20 and 30 hours.
 63. A process according to any one of claims 49 to 62 wherein the temperature of the reaction in step d is maintained between 20 and 26° C.
 64. A process according to claim 53 where the unreacted aldehydes are capped by the addition of about 2 molar equivalents of NaBH₄.
 65. A process according to any one of claims 49 to 64 further comprising a step of purifying the polysaccharide:carrier protein conjugate.
 66. A process according to any one of claims 49 to 65 further comprising a step of purifying the polysaccharide:carrier protein conjugate by tangential flow filtration and diafiltration.
 67. An immunogenic conjugate comprising Streptococcus pneumoniae serotype 10A polysaccharide covalently linked to a carrier protein obtained by the process of any one of claims 49 to
 66. 68. An immunogenic conjugate comprising Streptococcus pneumoniae serotype 10A polysaccharide covalently linked to a carrier protein obtainable by the process of any one of claims 49 to
 66. 69. An immunogenic conjugate according to claim 67 or 68, where said conjugate has a molecular weight between 500 and 15000; 500 and 10000; 2000 and 10000; or 3000 and 8000 kDa.
 70. An immunogenic conjugate according to claim 67 or 68, where said conjugate has a molecular weight between 3000 and 8000 kDa.
 71. An immunogenic conjugate according to any one of claims 67 to 70, where said conjugate comprises less than about 50, 45, 40, 35, 30, 25, 20 or 15% of free serotype 10A polysaccharide compared to the total amount of serotype 10A polysaccharide.
 72. An immunogenic conjugate according to any one of claims 67 to 70, where said conjugate comprises less than about 20% of free serotype 10A polysaccharide compared to the total amount of serotype 10A polysaccharide.
 73. An immunogenic conjugate according to any one of claims 67 to 72, where the ratio of serotype 10A polysaccharide to carrier protein in the conjugate is between 0.5 and
 3. 74. An immunogenic conjugate according to any one of claims 67 to 72, where the ratio of serotype 10A polysaccharide to carrier protein in the conjugate is between 0.8 and 1.4.
 75. An immunogenic conjugate according to any one of claims 67 to 74, where at least 40% of said immunogenic conjugate has a Kd below or equal to 0.3 in a CL-4B column.
 76. An immunogenic conjugate according to any one of claims 67 to 74, where between 50% and 80% of the serotype 10A immunogenic conjugate has a Kd below or equal to 0.3 in a CL-4B column.
 77. An immunogenic conjugate according to any one of claims 67 to 76, where the degree of conjugation of said immunogenic conjugate is between 2 and 15, 2 and 13, 2 and 10, 2 and 8, 2 and 6, 2 and 5, 2 and 4, 3 and 15, 3 and 13, 3 and 10, 3 and 8, 3 and 6, 3 and 5, 3 and 4, 5 and 15, 5 an 10, 8 and 15, 8 and 12, 10 and 15, or 10 and
 12. 78. An immunogenic conjugate according to any one of claims 67 to 76, where the degree of conjugation of said immunogenic conjugate is between 6 and
 8. 79. An immunogenic conjugate comprising Streptococcus pneumoniae serotype 22F polysaccharide covalently linked to a carrier protein obtained by the process of any one of claims 49 to
 66. 80. An immunogenic conjugate comprising Streptococcus pneumoniae serotype 22F polysaccharide covalently linked to a carrier protein obtainable by the process of any one of claims 49 to
 66. 81. An immunogenic conjugate according to claim 79 or 80, where said conjugate comprises at least 0.5, 0.6 or 0.7 mM acetate per mM serotype 22F polysaccharide.
 82. An immunogenic conjugate according to claim 79 or 80, where the ratio of mM acetate per mM serotype 22F polysaccharide in the immunogenic conjugate to mM acetate per mM serotype 22F polysaccharide in the isolated polysaccharide is at least 0.7.
 83. An immunogenic conjugate according to any one of claims 79 to 82, where the ratio of mM acetate per mM serotype 22F polysaccharide in the immunogenic conjugate to mM acetate per mM serotype 22F polysaccharide in the activated polysaccharide is at least 0.7.
 84. An immunogenic conjugate according to any one of claims 79 to 83, where said conjugate has a molecular weight between 400 and 15000; 500 and 10000; 2000 and 10000 kDa; 3000 and 8000 kDa; or 3000 and 5000 kDa.
 85. An immunogenic conjugate according to any one of claims 79 to 83, where said conjugate has a molecular weight between 3000 and 5000 kDa.
 86. An immunogenic conjugate according to any one of claims 79 to 85, where said conjugate comprises less than about 50, 45, 40, 35, 30, 25, 20 or 15% of free serotype 22F polysaccharide compared to the total amount of serotype 22F polysaccharide.
 87. An immunogenic conjugate according to any one of claims 79 to 83, where said conjugate comprises less than about 20% of free serotype 22F polysaccharide compared to the total amount of serotype 22F polysaccharide.
 88. An immunogenic conjugate according to any one of claims 79 to 87, where the ratio of serotype 22F polysaccharide to carrier protein in the conjugate is between 0.5 and 2,
 89. An immunogenic conjugate according to any one of claims 79 to 87, where the ratio of serotype 22F polysaccharide to carrier protein in the conjugate is between 0.8 and 1.2.
 90. An immunogenic conjugate according to any one of claims 79 to 89, where at least 40% of said immunogenic conjugate has a Kd below or equal to 0.3 in a CL-4B column.
 91. An immunogenic conjugate according to any one of claims 79 to 89, where between 50% and 80% of the serotype 22F immunogenic conjugate has a Kd below or equal to 0.3 in a CL-4B column.
 92. An immunogenic conjugate according to any one of claims 79 to 91, where the degree of conjugation of said immunogenic conjugate is between 2 and 15, 2 and 13, 2 and 10, 2 and 8, 2 and 6, 2 and 5, 2 and 4, 3 and 15, 3 and 13, 3 and 10, 3 and 8, 3 and 6, 3 and 5, 3 and 4, 4 and 7, 5 and 15, 5 an 10, 8 and 15, 8 and 12, 10 and 15, or 10 and
 12. 93. An immunogenic conjugate according to any one of claims 79 to 91, where the degree of conjugation of said immunogenic conjugate is between 4 and
 7. 94. An immunogenic conjugate comprising Streptococcus pneumoniae serotype 33F polysaccharide covalently linked to a carrier protein obtained by the process of any one of claims 49 to
 66. 95. An immunogenic conjugate comprising Streptococcus pneumoniae serotype 33F polysaccharide covalently linked to a carrier protein obtainable by the process of any one of claims 49 to
 66. 96. An immunogenic conjugate according to claim 94 or 95, where said conjugate comprises at least 0.5, 0.6 or 0.7 mM acetate per mM serotype 33F polysaccharide.
 97. An immunogenic conjugate according to claim 94 or 95, where the ratio of mM acetate per mM serotype 33F polysaccharide in the immunogenic conjugate to mM acetate per mM serotype 33F polysaccharide in the isolated polysaccharide is at least 0.7.
 98. An immunogenic conjugate according to any one of claims 94 to 97, where the ratio of mM acetate per mM serotype 33F polysaccharide in the immunogenic conjugate to mM acetate per mM serotype 33F polysaccharide in the activated polysaccharide is at least 0.7.
 99. An immunogenic conjugate according to any one of claims 94 to 98, where said conjugate has a molecular weight between 500 and 30000; 500 and 25000; 500 and 20000; 500 and 15000; 500 and 10000; 1000 and 10000; 1000 and 8000; 1000 and 5000; 2000 and 10000 kDa; 2000 and 8000; or 2000 and 5000 kDa.
 100. An immunogenic conjugate according to any one of claims 94 to 98, where said conjugate has a molecular weight between 1000 and 5000 kDa.
 101. An immunogenic conjugate according to any one of claims 94 to 100, where said conjugate comprises less than about 50, 45, 40, 35, 30, 25, 20 or 15% of free serotype 33F polysaccharide compared to the total amount of serotype 33F polysaccharide.
 102. An immunogenic conjugate according to any one of claims 94 to 100, where said conjugate comprises less than about 20% of free serotype 22F polysaccharide compared to the total amount of serotype 22F polysaccharide.
 103. An immunogenic conjugate according to any one of claims 94 to 102, where the ratio of serotype 33F polysaccharide to carrier protein in the conjugate is between 0.5 and
 2. 104. An immunogenic conjugate according to any one of claims 94 to 102, where the ratio of serotype 22F polysaccharide to carrier protein in the conjugate is between 0.5 and 1.5.
 105. An immunogenic conjugate according to any one of claims 94 to 104, where at least 40% of said immunogenic conjugate has a Kd below or equal to 0.3 in a CL-4B column.
 106. An immunogenic conjugate according to any one of claims 94 to 104, where between 40% and 80% of the serotype 33F immunogenic conjugate has a Kd below or equal to 0.3 in a CL-4B column.
 107. An immunogenic conjugate according to any one of claims 94 to 106, where the degree of conjugation of said immunogenic conjugate is between 2 and 15, 2 and 13, 2 and 10, 2 and 8, 2 and 6, 2 and 5, 2 and 4, 3 and 15, 3 and 13, 3 and 10, 3 and 8, 3 and 6, 3 and 5, 3 and 4, 5 and 15, 5 an 10, 8 and 15, 8 and 12, 10 and 15, or 10 and
 12. 108. An immunogenic conjugate according to any one of claims 94 to 106, where the degree of conjugation of said immunogenic conjugate is between 4 and
 6. 109. An immunogenic conjugate according to any one of claims 67 to 108 where the carrier protein is CRM₁₉₇.
 110. A process according to any one of claims 49 to 66 where the carrier protein is CRM₁₉₇.
 111. A process according to any one of claims 49 to 66 where the activated polysaccharide is of serotype 10A.
 112. A process according to any one of claims 49 to 66 where the activated polysaccharide is of serotype 22F.
 113. A process according to any one of claims 49 to 66 where the activated polysaccharide is of serotype 33F.
 114. A process according to anyone of claims 49 to 66 and 110 to 113 wherein said process further comprises the step of formulating the conjugate in a multivalent vaccine.
 115. An immunogenic composition comprising an immunogenic conjugate according to any one of claims 67 to 109 and a physiologically acceptable vehicle.
 116. An immunogenic composition according to claim 115 further comprising at least one additional antigen.
 117. An immunogenic composition according to claim 115 or 116 further comprising an adjuvant.
 118. An immunogenic composition according to claim 117 where the adjuvant is aluminium phosphate.
 119. A vaccine comprising an immunogenic composition according to any one of claims 115 to
 118. 120. A method of treating or preventing a Streptococcus pneumoniae infection, disease or condition associated with serotype 10A, 22F or 33F Streptococcus pneumoniae in a subject, the method comprising the step of administering a therapeutically or prophylactically effective amount of an immunogenic composition according to any one of claims 115 to 118, or the vaccine of claim
 119. 